Complement depletion using recombinant human C-3 derivatives

ABSTRACT

The invention provides isolated polypeptides having complement-modulating activity. Specifically, the invention resides in the provision of isolated polypeptides having complement depleting properties, i.e. that effect an efficient consumption of complement in human serum. The current invention thus provides human C3 derivatives that are capable of forming C3 convertases exerting an extended CVF, Bb-like half-life of up several hours, compared to 1.5 minutes of the naturally occurring C3 convertases, thus escaping the physiological degradation mechanisms.

This application is a continuation of U.S. patent application Ser. No.10/884,813, filed on Jul. 2, 2004, now U.S. Pat. No. 7,553,931 whichclaims the benefit of U.S. Provisional Application No. 60/484,797, filedJul. 3, 2003, and entitled Complement Depletion using recombinant HumanC3-Derivatives, and which is hereby incorporated herein by reference inits entirety.

BACKGROUND OF THE INVENTION

The activation of the complement system can be achieved by threedifferent pathways, the classical antibody-dependent activation pathway,the alternative activation pathway and the lectin-activation pathway.The alternative activation pathway as well as the lectin-pathway wereshown not to be dependent on antibodies. All pathways share a similarcascade-like organization, wherein a protease acts on zymogenes of asubsequent protease. This cascade results in an amplification of theinitiation signals. The central step of the complement cascade residesin the formation of a C3-convertase, which cleaves C3 to C3b and C3a(FIG. 1). Subsequently, the resulting C3b can act as a part of aC5-convertase, which cleaves C5 in C5b and C5a. In the terminal pathway,the gradual accumulation of C6, C7, C8 and several molecules C9 resultsin the formation of the membrane attack complex which is capable offorming a pore in the membrane of the target cells, thereby effectinglysis of the cells.

The complement protein C3 is the central component of all activationpathways. It is predominantly expressed in the liver as a 1663 aminoacid precursor protein (Alper et al., 1969). After the 22 amino acidsignal sequence has been cleaved off, the precursor protein isproteolytically cleaved into two chains by removal of four arginineresidues. The resulting α-chain has a molecular weight of 115 kDa andthe β-chain has a molecular weight of 73 kDa (DeBruijn and Fey, 1985).The chains are linked by a disulfide bridge and by non-covalentinteractions (Dolmer and Sottrup-Jensen, 1993; Janatova, 1986).Furthermore, the resulting 188 kDa protein carries a carbohydrate chainon each chain, which consists of 5 to 9 mamose residues and twoN-acetylglucosamine residues (Hirani et al., 1986).

C3 is cleaved between the amino acids Arg⁷²⁶ and Ser⁷²⁷ by theC3-convertases. The 9 kDa C3a, which results from the cleavage, is ananaphylatoxin and causes an increase in chemotaxis as well as anincrease in the permeability of the blood capillaries. By cleavage ofthe 179 kDa-C3b between the amino acid Cys⁹⁸⁸ and Glu⁹⁹¹ a highlyreactive thioester is released, by the use of which C3b binds on thecell surfaces via transacetylation (Tack et al., 1980). Furthermore,several binding sites for different complement proteins are exposed bythe cleavage, which explains the various interactions of the C3bmolecule. Several regulatory complement proteins interact with C3b,which comprises binding sites for CR1 or Factor H, which act as cofactors for the cleavage by Factor I. Factor I cleaves C3b betweenArg¹²⁸¹ and Ser¹²⁸², and Arg1298 and Ser¹²⁹⁹, whereby the fragments C3fand C3bi emerge, the latter of which is inactive and unable to bindFactor B and C5 (Lachmann et al., 1982; Davis et al., 1982). C3bi,however, is capable to remain on the surface of pathogens, where it isrecognized by CR3, which occurs on macrophages and killer cells.Subsequently, CR3 mediates the destruction of pathogens (Newman et al.,1984). In case CR1 acts as a cofactor for the protease, Factor I canadditionally cleave between amino acids Arg⁹³² and Glu⁹³³, therebyforming C3dg and C3c (Ross et al., 1982). C3dg is also capable to remainon the surface and is recognized by CR2 (CD21), which is expressed onB-lymphocytes and dendritic cells (Law and Dodds, 1997). The binding ofC3dg to the complement receptor CR2 leads to the activation of B cells(Bohnsack and Cooper, 1988).

After the degradation of C3 by a protease from the venom of the cobraNaja siamensis the fragment C3o is formed, which no longer contains theamino acids 730-739. However, C3o is capable of binding Factor B(O'Keefe et al., 1988). In contrast, the cleavage product of the FactorI proteolysis C3o cannot form a convertase. Based on the comparison ofC3c and C3o, one region in C3o of the amino acid sequence ⁹³³EGVQKEDIPPappeared to be responsible for binding to Factor B. In further studies,the amino acids ⁹³⁷KED were mutated to alanine. However, no changes inthe binding characteristics of Factor B to C3b could be shown(Taniguchi-Sidle und Isenman, 1994).

Activated complement proteins cannot distinguish between externalsubstances and substances which occur naturally in the body. Thereby, itis ensured that for example self-reactive B-cells can be eliminated.Thus, a plurality of regulatory mechanisms is necessary for protectinghealthy cells which occur naturally in the body.

The regulation is effected by short half life of the activatedcomplement proteins on the one hand and by plasma proteins such as theC1-Inhibitor (C1-Inh), Factor H and Factor I, as well as membrane-boundproteins such as the Decay-Accelerating-Factor (DAF, CD55), theMembrane-Cofactor-Protein (MCP, CD46) and the Complement Receptor 1(CR1, CD35) on the other hand, which regulate the complement cascade onspecific levels.

C1-Inh controls the activation of C1 by binding to activated C1r and C1swhich results in the dissociation of C1q. The time period for thecleavage of C2 and C4 by activated C1 is restricted to a few minutes byC1-Inh (Mollnes und Lachmann, 1988). The C4 binding protein (C4bp) bindsto C4b and separates it from C2b. Additionally, it acts as a co-factorfor the cleavage of C4b and C3b by Factor I (Scharfstein et al., 1978).The C3-convertase of the classical pathway is inactivated in the samemanner by DAF, which exists on all peripheral cells of the blood,epithel and endothel (Lublin and Atkinson, 1989, Lublin and Atkinson,1990).

C3b represents the central component of all three activation pathways.C3b is regulated by Factor H, CR1, DAF as well as by MCP. Here, Bb iscompetitively displaced by CR1, Factor H and DAF from the complex of theC3-convertase C3bBb (Makrides et al., 1992). Subsequently, C3b iscleared by Factor I and inactivated (Pangburn and Müller-Eberhard,1984). MCP directly attacks C3b and is also a co-factor for the cleavageby Factor I. Protectin (CD95) is a further membrane-bound regulatoryprotein. It inhibits the polymerization of C9 by binding to C8 and C9(Mollnes and Lachmann, 1988).

Besides the regulation for the activation, an additional transcriptionalcontrol of the complement genes exists. For example, several genes ofthe complement proteins are upregulated by cytokine andIFN_(γ)-activated transcription factors after damaging a tissue(Volanakis, 1995).

The strict regulatory mechanisms prevent an attack of the complementsystem on cells which occur naturally in the body. However, body tissuecan be damaged by unregulated activation triggered by diverse diseases.In this situation, the activation of the complement is not the primaryreason for disease. However, the resulting damaging of the tissue ismediated by the complement. Diseases which are connected with theactivation of the complement can be divided into three groups: Chronicaldiseases, acute diseases and incompatibility towards biomaterials. Thegroup of acute diseases comprise for example asthma (Regal et al., 1993;Regal and Fraser, 1996), sepsis (Hack et al., 1989; Hack et al., 1992),hyperacute rejection in connection with transplantations orxenotransplantations (Bach et al., 1995; Baldwin et al., 1995),pneumonia (Eppinger et al., 1997) and cardiac infarction (Kilgore etal., 1997), as well as a massive C3a-accumulation, which occurs inconnection with the cardiopulmonale bypass-operation (Kirklin et al.,1983; Homeister et al., 1992). The chronical diseases comprise, forexample, systemic lupus erythematodes (SLE) (Belmont et al., 1986; Buyonet al., 1992), glomerulonephritis (Couser et al., 1985; Couser et al.,1995), rheumatoide arthritis (Kemp et al., 1992; Wang et al., 1995),Alzheimer's disease (Rogers et al., 1992; Morgan et al., 1997),myastenia gravis (Lennon et al., 1978; Piddlesden et al., 1996) andmultiple sclerosis (Piddlesden et al., 1994; Williams et al., 1994) aswell as organ rejection after transplantations or xenotransplantations(Baldwin et al., 1995; Dalmasso, 1997). The group of incompatibilitiestowards biomaterials was described in connection with operation materialat a cardiopulmonal bypass (Craddock et al., 1977; Mollnes, 1997), withdepositions of blood platelets (Gyongyossy-Issa et al., 1994) and withconducting hemodialysis (Cheung et al., 1994; Mollnes, 1997).

A reduced protein concentration of a complement protein or mutationswhich lead to a total loss of the protein are the reason for manycomplement-associated diseases. Factor I-deficiency results in a verysmall content of C3 and other complement proteins of the cascade in theblood. This leads to diverse diseases, such as a monthly occuringmeningitis which is associated with menstruation (Gonzales-Rubio et al.,2001). Factor H-deficiency by gene mutation is associated with thehemolytic-uremic syndrom (Zipfel et al., 2001). An unrestricted activityin the classical activation by depletion of C1, C2 or C4 leads forexample to a higher disposition towards systemic lupus erythematodes(Morgan and Walport, 1991). A depletion of a component from thealternative activation such as Factor B or Factor D leads to a highersusceptibility towards infections (Morgan and Walport, 1991).

Complement-associated diseases occur both with an increased anddecreased complement activation. In case the regulation is disturbed orthe activation is prevented, effective complement modulators are needed.

The group of complement inhibitors for therapeutic use comprisesproteins such as the C1-Inhibitor and the soluble complement regulatorssCR1 (soluble CR1), sMCP or sDAF, antibodies against C5 or C3 andsmaller molecules such as the peptide Compstatin or RNA-aptamers.Several complement inhibitors are tested in clinical phases I, II orIII, such as the C5-Inhibitor Pexelizumab, a monoclonal antibody for useat cardiopulmonal bypass (Whiss, 2002) or the soluble complementreceptor sCR1 (Zimmerman et al., 2000).

The C1-Inhibitor is the only plasma protein which has been tested in invivo-studies (Struber et al., 1999; Horstick, 2002). The serine proteaseis a suicide inhibitor of the serpine family which inhibits activatedC1s and C1q by binding to the active site (Sim et al., 1979). Thedisadvantages of these molecules relate to the sole inhibition of theclassical activation pathways as well as in the susceptibility of theprotein towards the inactivation by elastase. For this reason,elastase-resistent C1-Inhibitor mutants were generated (Eldering et al.,1993).

The recombinant complement inhibitors embrace soluble regulators such assCR1, sMCP and sDAF (Christiansen et al., 1996). The soluble complementreceptor sCR1 its as C3- and C5-convertase-inhibitor and has been testedsuccessfully in diverse animal models such as for myasthenia gravis(Piddlesden et al., 1996), multiple sclerosis (Piddlesden et al., 1994)or asthma (Regal et al., 1993). By altering the condition of expression,it was possible to increase the short half-life of approx. 8 h in vivoup to 70 h. It is proposed that a different glycosylation pattern isresponsible for the increased half-life (Weismann et al., 1990;Zimmerman et al., 2000).

The complement receptors MCP and DAF act as complement inhibitors bothin vitro and in vivo, for example in the model of reverse passiveArthus-reaction (Moran et al., 1992; Christiansen et al., 1996). sDAFaccellerates the decomposition of both the classical and the alternativeC3- and C5-convertases. However, sDAF does not act as a co-factor forthe cleavage of Factor I (Kinoshita et al., 1985). In contrast, sMCPacts as cofactor for the cleavage of C3b and C4b by Factor I. However,it does not act on the convertases (Liszewski and Atkinson, 1992).

Protectin (CD59) is a further membrane protein which protects naturallyoccuring cells of the body from MAC-mediated damage. It binds to C5b-8and prevents the formation of a pore in the membrane by binding of C9(Davies, 1996). Its soluble counterpart, sCD59, showed inhibition invitro (Sugita et al., 1994).

A further group of complement inhibitors consists of antibodies, whereinC5 in particular represents an attractive target protein, since itsconcentration in the serum is clearly lower than the one of C3.Monoclonal antibodies combine the advantage of specifity and highaffinity with a relatively long half-life and the ease of production inlarge amounts. One prerequisite for the therapeutic application is thehuman origin of the antibodies which prevents an immune response, forexample the human anti-mouse-antibody-response. Several antibodiesagainst C3 (Kemp et al., 1994), C3a (Burger et al., 1988; Elsner et al.,1994) or against C5a (Ames et al., 1994; Park et al., 1999) have beendeveloped. Some have been tested in different animal models, for examplefor nephritis (Wang et al., 1996), collagen-induced arthritis (Wang etal., 1995), myocardial ischemia und reperfusion (Vakeva et al., 1998).

Anaphylatoxin-receptor-antagonists (Konteatis et al., 1994; Pellas etal., 1998; Heller et al., 1999) and RNA-aptamers, which inhibit theC5-cleavage (Biesecker et al., 1999) belong to the group of complementinhibitors with low molecular weight. Compstatin, a C3-Inhibitor, bindsto native C3 and prevents its cleavage in C3b. By application ofCompstatin, the hyperacute rejection of transplants in an ex-vivopig-to-human-liver-transplantation was prevented (Fiane et al., 1999a;Fiane et al., 1999b).

Cobra Venom Factor (CVF) is a potent complement modulator of naturalorigin. CVF is a 149 kDa-glycoprotein from the venom of the cobraspecies Naja, Ophiophagus and Hemachatus (Müller-Eberhard andFjellström, 1971). The nontoxic protein consists of three chains, the 68kDa α-chain, the 48 kDa β-chain and the 32 kDa γ-chain which are linkedby disulfide bridges. Additional intramolecular disulfide bridges existboth in the α- and β-chain (one in the chain and six in the β-chain;Vogel et al., 1996). The γ-chain can exhibit different sizes due todifferent processing on the C-terminus (Vogel and Müller-Eberhard,1984). Two carbohydrate residues are attached to the α-chain and one tothe β-chain in form of complex, N-bound oligosaccharide chains (Vogeland Müller-Eberhard, 1984; Grier et al., 1987).

The percentual composition of the secondary structure of CVF wasdetermined by circular dichroism. The composition shows a high analogyto the composition of the secondary structures of the human three-chainC3-derivate C3c. For CVF 11% helices, 47% βsheets and 18% β-loops weredetermined. The C3c-molecule also has 11% helices and 47% β-sheets. Incontrast, human C3 consists of 24% helices and 32% β-sheets (Vogel etal., 1984). In the primary structure of the pre-pro-CVF the α-chain isencoded first, followed by the γchain and subsequently by the β-chain.On the C-terminus of the α-chain 4 arginine residues are located,followed by aC3a-homologous region. Subsequent to the γ-chain, aC3d-homologous region is located. Both the signal peptide and thearginine residues and the C3a and C3d-homologous regions are removedpost-translationally, thereby generating the three-chain structure. Thevenom protease, which is thought to be responsible for the modificationalso cleaves C3 in a CVF-similar structure (O'Keefe et al., 1988).

CVF shares an identity of 85% and a similarity of 92% on the proteinlevel with cobra C3 (coC3). With human C3 the identity amounts to 51%and the similarity to 70% (Fritzinger et al., 1992; Fritzinger et al.,1994; Vogel et al., 1996). Moreover, both proteins have a chainstructure of the same kind.

This high similarity is also reflected by the fact that CVF—as C3b—canbind to Factor B and forms a convertase by the Factor D initiatedcleavage of B in Bb and Ba. In contrast to C3Bb, the CVF dependentconvertase CVFBb is a C3- and C5-convertase. By the resistence of CVFBbtowards Factor H and Factor I, a convertase is formed with a much higherhalf-life of 7 h (Vogel and Müller-Eberhard, 1982) under physiologicalconditions. In comparison, C3bBb has a half-life of 1.5 min (Medicus etal., 1976).

In addition to the increased stability, the CVF dependent convertaseCVFBb cleaves C3 and C5 also in fluid phase, whereas the dependentconvertase C3bBb is only active when bound to the cell surface (Vogel etal., 1996). CVF unifies all the above characteristics and leads to apermanent activation of the complement system and to decouplementationresulting thereof.

The decomplementing characteristic of CVF offers a variety ofapplications. After decomplementation the synthesis of complementprotein takes approx. 7 days; during this time e.g. the function of thecomplement system in the immune response in vivo as well as in thepathogenesis of diseases can be studied (Cochrane et al., 1970; Ryan etal., 1986).

In various xenotransplantation models, such as liver transplantationfrom guinea pigs to rats, heart transplantation from hamsters to mice aswell as islet cell transplantations from rats to mice, CVF wassuccessfully employed (Chrupcala et al., 1994; Chrupcala et al., 1996;Lin et al., 2000; Oberholzer et al., 1999). In all these cases,hyperacute rejection of transplant could be prevented by CVF. Differentstudies demonstrate that also for diseases like arthritis (Lens et al.,1984), arteriosclerosis (Pang and Minta, 1980) and encephalomyelitis(Morariu and Dalmasso, 1978) CVF can be therapeutically employed.

The problem with therapeutic applications of CVF, however, predominantlyresides in the strong immunogenic character of CVF. CVF contains aforeign peptide structure and complex, Nbound oligosaccharide chainswith terminal galactosyl residues, which have a significant immunogenicpotential (Taniguchi et al., 1996). Consequently, CVF is not suitablefor repetitive application. With a relative high portion of carbohydratestructures (7.4%, Vogel and Müller-Eberhardt, 1984) CVF differs clearlyfrom human C3 which only has 1.7% (Hirani et al., 1986). Activityanalyses in complement consumption-assays and bystander lysis-assays ofCVF deglycosylated by n-glycanase showed that the oligosaccharide chainsof CVF are not necessary for both C3-convertase and C5-convertaseactivity. A reduction of the immunogenicity, however, cannot be achievedby deglycosylation since deglycosylated CVF is still stronglyimmunogenic due to its foreign amino acid composition.

In an attempt to reduce the immunogenicity of CVF, the CVF α-chain wasreplaced by the corresponding human C3-β-chain (Kölln et al., 2001). Theresulting hybrid protein, however, is still strongly immunogenic andthus inappropriate for therapeutic uses.

From these published results, no information is available which couldaid in designing a human C3-derivative i) capable of forming a stableC3-convertase comparable to CVFBb, ii) and suitable for therapeuticapplications. Furthermore, published CVF/cobra C3 hybrids (Wehrhahn,2000) are not suitable to provide any valuable data with regard to thetertiary structure of a C3-derivative required for effective binding ofFactor B and for increasing the half life of the resultingC3-convertase.

The identity of cobra C3 with human C3 is too low to allow any specificstructural conclusions.

Accordingly, there exists a need to identify polypeptides that exhibitcomplement-depleting activity and to develop methods of preparing thesecompounds recombinantly as therapeutics. There also exists a need toidentify polypeptides having reduced or eliminated immunogenicity, whichpolypeptides can be used therapeutically for treatingcomplement-associated disorders and disorders affected by complementactivation, respectively.

The present invention satisfies this need, and provides relatedadvantages as well.

SUMMARY OF THE INVENTION

The invention provides isolated polypeptides having complementmodulating activity. Specifically, the invention resides in theprovision of isolated polypeptides having complement-depletingproperties, i.e. that effect an efficient consumption of complement inhuman serum. Thus, the invention provides molecules capable ofeffectively inhibiting the complement system by depletion.

More specifically, the invention relates to polypeptides which arederivatives of the human complement component C3 (referred to herein as‘human C3’, or ‘C3’, respectively), where the carboxy terminal (Cterminal) part of the polypeptide is replaced by a carboxyterminal partor fragment of Cobra Venom Factor (CVF). The amino acid sequences ofhuman C3 and CVF are depicted as SEQ ID NO:2 and SEQ ID NO:4,respectively.

The number of amino acids of CVF which replace the C terminal C3fragment is either equal or less than the number of amino acids whichhave been ‘removed’ in relation to the native human C3 sequence. Thepolypeptides of the invention, which are in some respect hybridpolypeptides of C3 and CVF or chimeric proteins, respectively, requireat least the presence of amino acids 1575 to 1617 of the CVF sequenceshown as SEQ ID NO:4, in order to retain complement depleting activity.

In addition, without departing from the spirit of the invention, it maybe desired for specific purposes, however, to attach additional non-C3and non-CVF amino acids to the carboxy terminus of the hybrid proteins.

The polypeptides of the invention being at least 70% identical to thesequence of human C3 are less immunogenic than CVF (SEQ ID NO:4) or CVFin which the α-chain is replaced by the corresponding human C3 β-chain.

Also provided are methods of modulating or depleting complement in ahuman subject and methods of therapeutical treatment, respectively, byadministering to the human an effective amount of a C3-derivative of theinvention.

A further embodiment is a pharmaceutical composition comprising aprotein of the invention.

The invention also provides nucleic acid molecules encoding novelC3-derivatives.

Vectors comprising nucleic acid molecules encoding novel C3-derivativesand isolated host cells transfected with said vectors are provided aswell.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows an alignment of the amino acid sequences of humancomplement component C3 (C3) and of Cobra Venom Factor (CVF). (‘*’indicates identical amino acids; ‘:’ indicates conservative amino acidreplacements; ‘.’ indicates semi-conservative amino acid replacementsaccording to Clustal W).

FIG. 2 shows schematic representations of C3, pre-CVF, of construct H2according to Kölln et al. (2001) and of various C3-derivatives accordingto the present invention. CVF and CVF parts within hybrid constructs areshown in white, C3 and C3 parts are dark coloured. The AA values aboveconstructs H5, H6, H6 truncated, respectively, indicate the number ofCVF amino acids contained in the expressed hybrid proteins. For twofurther constructs (H7 and H7 truncated), the C3 part has been extendedto the 3′ end, thus encoding for 50 additional C3 amino acids, whereasdifferent lengths are used for the CVF part. The construct H7 truncatedrepresents a specific embodiment of the invention, where the hybridprotein comprises the minimum number of 43 CVF amino acids (amino acids1575 to 1617 of SEQ ID NO:4).

FIG. 3 shows a simplified map of pUC18CVF* and pcDNACVF.

FIG. 4 shows a simplified map of pUC18hC3 and pcDNA3hC3.

FIG. 5 is a schematic representation of the Construct H5 encoding ahybrid protein of approx. 91% identity with human C3A: Chain structuresof C3 and pre-CVF. B: Structure of the cDNA of H5. The dark part of thenucleic acid from the 5′ end up to the BglII cleavage site representspart of the molecule comprising C3 nucleic acids. the white partdownstream of the BglII site comprises CVF nucleic acids.

FIGS. 6(A+B)shows the cloning strategy used for generating construct H5expressing a hybrid protein of 1663 amino acids in length, which aminoterminally comprises amino acids 1 to 1388 of C3 followed by amino acids1368 to 1642 of CVF at the carboxy terminus.

FIG. 7 shows a solid phase assay with St-H5, St-CVF and St-C3. Thecomplement-consumption-assay was conducted in an ELISA plate. Theproteins which were immobilized to Strep-Tactin were incubated withhuman serum at 37° C. in an incubation shaker at 150 rpm for 3 h.Subsequently, the reaction mixtures were transferred to 2 ml reactiontubes. 100 μl GVBS⁺⁺ and 30 μl sensitized sheep erythrocytes (5×10⁸cells/ml) were added to the mixture. Then, the mixture was furtherincubated in a thermomixer until the samples with serum alone reached ahemolysis of approx. 80% compared to the controls with ddH₂O. Afteraddition of 850 μl GVBS⁺⁺ the mixture was centrifuged (4° C., 2000 xg, 2min) and the supernatants were measured at 412 nm. The Figure shows themean values±standard deviation of at least three independentexperiments.

FIG. 8 shows the complement-consuming activity of CVF, St-H5 and C3. Thesamples (40 μl) were incubated with human serum (approx. 10 μl) at 37°C. in a thermomixer under agitation for 3 h. Subsequently, 100 μl GVBS⁺⁺and 30 μl sensitized sheep-erythrocytes (5×10⁸ cells/ml) were added andthe mixture was further incubated in a thermomixer until the serumcontrols reached hemolysis of approx. 80% compared to the control withddH₂O. After addition of 850 μl GVBS⁺⁺ the mixture was centrifuged (4°C., 2000 xg, 2 min) and the supernatants were measured at 412 nm. Thefigure shows the mean values±standard deviation of at least threeindependent experiments.

FIG. 9 shows functional characteristics of the H5 dependent C3convertase.

(A) Activation of purified human factor B. Purified hC3 (lane 1), CVF(lane 3) and H5 (lane 6) were incubated for 2 h in the presence offactor D, factor B, and Mg²⁺. Inhibition of convertase formation by EDTA(hC3: lane 2; nCVF: lane 4) and incubation of factor B with factor D andMg²⁺ (lane 5) were performed as controls. After separation of thereaction mixture by 10% SDS-PAGE under non-reducing conditions,generation of the cleavage products Bb and Ba was analyzed by westernblotting using anti-factor B antibodies.

(B-D) Catalytic activity of nCVFBb, hC3Bb, and H5Bb complexes. Shown isthe time-dependent generation of fluorescent amido-methylcoumarin (AMC)from Boc-Leu-Gly-Arg-AMC by the different C3 convertases (B: nCVFBb; C:hC3Bb; D: H5Bb) after preincubation at 37° C. for 60 min (squares), 120min (triangles), 180 min (dots), and 240 min (crosses) in the absence ofthe fluorogenic substrate. New formation of convertase complexes wasinhibited by addition of EDTA prior to preincubation. Convertaseactivity of complexes without preincubation (diamonds) was used ascontrol. After addition of the fluorogenic peptide to the samples, thetime dependent release of AMC was followed by measuring fluorescence at465 nm. (E) Stability of nCVFBb and H5Bb complexes. Based on the slopesin FIG. 9B, D, the catalytic activity of both C3 convertases (nCVFBb:gray triangles; H5Bb: black squares) was determined. Shown are meanvalues±s.d. obtained from at least three independent experiments.

FIG. 10 shows a comparison of the amino acid residues of human C3 andCVF at the C-terminal regions. The restriction sites for the cloning ofH5 (BglII) and H6 (Bsp1407I) are indicated. (* identical amino acids, :conservative amino acid exchange, . semi-conservative amino acidexchange; classification according to Clustal W).

FIG. 11 shows a schematic representation of the construct H6. A: Chainstructures of C3 and pro-CVF. B: Structure of H6. cDNA.

FIG. 12 shows a schematic representation of the cloning strategy for H6.

FIG. 13 shows a solid phase-assay with St-H6, St-CVF and St-C3. Therecombinant proteins were bound to Strep-Tactin which in turn wasimmobilized on the ELISA plate. Then, a solid phase assay was performedin the ELISA plate. The samples were incubated with human serum at 37°C. in an incubation shaker at 150 rpm for 3 h. Subsequently, thereaction mixtures were transferred to 2 ml reaction tubes. 100 μl GVBS⁺⁺and 30 μl sensitized sheep-erythrocytes (5×10⁸ cells/ml) were added.Then, the mixture was incubated in a thermomixer until the serumcontrols reached a hemolysis of approx. 80% compared to the control withddH₂O. After addition of 850 μl GVBS⁺⁺ the mixture was centrifuged (4°C., 2000 xg, 2 min) and the supernatants were measured at 412 nm. Thefigure shows the mean values±standard deviation of at least threeindependent experiments.

FIG. 14 shows a complement-consumption-assay with CVF, His-H6 and C3.The samples (40 μl ) were incubated with human serum (approx. 10 μl) at37° C. in a thermomixer under agitation for 3 h. Subsequently, 100 μlGVBS⁺⁺ and 30 μl sensitized sheep-erythrocytes (5×10⁸ cells/ml) wereadded and the mixture was further incubated in a thermomixer until theserum controls reached a hemolysis of approx. 80% compared to thecontrol with ddH₂O. After addition of 850 μl GVBS⁺⁺ the mixture wascentrifuged (4° C., 2000 xg, 2 min) and the supernatants were measuredat 412 nm. The figure shows the mean values±standard deviation of atleast three independent experiments.

FIG. 15 shows the evaluation of the complement-consuming activity ofCVF, His-H6 and C3 by linear regression analysis.

FIG. 16 shows the results of a complement consumption assay with hybridsH5 and H6, CVF, and human C3. The samples (40 μl) were incubated withhuman serum (approx. 10 μl) at 37° C. in a thermomixer under agitationfor 3 h. Subsequently, 100 μl GVBS⁺⁺ and 30 μl sensitizedsheep-erythrocytes (5×10⁸ cells/ml) were added and the mixture wasfurther incubated in a thermomixer until the serum controls reached ahemolysis of approx. 80% compared to the control with ddH₂O. Afteraddition of 850 μl GVBS⁺⁺ the mixture was centrifuged (4° C., 2000 xg, 2min) and the supernatants were measured at 412 nm. The figure shows themean values±standard deviation of at least three independentexperiments.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides isolated polypeptides or proteins, respectively,that are biologically active derivatives of human complement componentC3, that are able to continuously activate human complement system inhuman serum and thus to temporarily achieve inactivation of thecomplement system.

As used herein, the term “human complement component C3” (‘human C3’ or‘C3’, respectively) refers to a polypeptide comprising the amino acidsequence of human complement component C3 shown in FIG. 1 (SEQ ID NO:2).

As used herein, the term ‘CVF’ refers to a polypeptide comprising theamino acid sequence of Cobra Venom Factor as known in FIG. 1 (SEQ IDNO:4).

As used herein, the terms “comprising,” “having,” “encoding,” and“containing,” and derivatives of these terms, are intended to beopen-ended. “consisting” is intended to be closed-ended.

The invention specifically relates to a polypeptide or protein having alength of 1638 to 1663 amino acids, which is a derivative of humancomplement component C3 (human C3), the amino acid sequence of which isshown in SEQ ID NO:2, wherein the carboxy terminal part of at least 68amino acid residues of human C3 is replaced by a partial sequence ofCobra Venom Factor (CVF), the amino acid sequence of which is shown inSEQ ID NO:4, which partial sequence comprises at least the 68 carboxyterminal amino acid residues of CVF or a fragment thereof lacking 1 to25 carboxy terminal amino acids, wherein said protein has at least 70%identity to human C3 or a fragment thereof, the fragment comprising atleast amino acids 1 to 1638 of SEQ ID NO:2. Thus, the invention providesa protein comprising a derivative of human complement. component C3(human C3), the human C3 having an amino acid sequence set forth as SEQID NO: 2, wherein the carboxy terminal part of at least 68 amino acidsof said human C3 is replaced by a partial sequence of Cobra Venom Factor(CVF), the CVF having an amino acid sequence set forth as SEQ ID NO: 4,wherein the partial sequence of CVF comprises at least at least 68carboxy terminal amino acids of CVF or a fragment thereof, said fragmentlacking 1 to 25 carboxy terminal amino acids, and wherein said proteinhas at least 70 percent identity to said human C3 or a fragment of saidhuman C3 comprising at least amino acids 1 to 1638 of the amino acidsequence set forth as SEQ ID NO: 2. An alignment of the C3 and CVFsequences is shown in FIG. 1.

In addition, without departing from the spirit of the invention, it maybe desired for specific purposes, however, to attach additional non-C3and non-CVF amino acids to the carboxy terminus of the hybrid proteins.

Decomplementing activity of C3/CVF hybrid proteins is observed withpolypeptides where the C3 α-chain was replaced by the correspondingcarboxy terminal amino acids of the CVF chain (including the γ- and theβ-chain of CVF). However, due to the high immunogenicity of suchpolypeptides, a higher degree of humanization is desired. Thus,according to a preferred embodiment, the polypeptides of the inventioncomprise an amino terminal C3 fragment containing the amino acidsforming the β-chain (amino acids 23 to 667 of SEQ ID NO:2) as well asadditional amino acids of the C3 chain following at the carboxy terminalend of the β-chain, i.e. from amino acid 668 towards the carboxyterminus of the peptide. At lest 68 amino acids of the C3 sequence arereplaced by amino acids of the corresponding CVF sequence. Reference ismade in this respect to FIG. 1, showing the alignment of the C3 and CVFsequences. The requirement that the amino acid sequence of thepolypeptides of the invention have at least 70% identity to the aminoacid sequence of human C3 is intended to ensure that immunogenicity ofthe hybrid proteins is kept at a relatively low level. The 70% valuethus also determines the minimum sequence stretch of the C3 sequencewhich is required for being combined with the amino acids of the CVFsequence which replace the corresponding carboxy terminal C3 aminoacids. It is desired to provide polypeptides where the identity with thehuman C3 sequence is at least 80%, or preferably at least 90% and mostpreferably at least 95%.

According to a preferred embodiment of the invention, the protein orpolypeptide, which is a derivative of human complement component C3(human C3), has an amino acid sequence, which is selected from the groupconsisting of:

a) the sequence shown in SEQ ID NO:6;

b) the sequence shown in SEQ ID NO:8;

c) the sequence shown in SEQ ID NO:10; and

d) the sequence shown in SEQ ID NO:12.

The most preferred constructs represented by SEQ ID NO:6 and SEQ ID NO:8are hereinafter also termed ‘H5’ and ‘H6’, respectively. The identity ofthe amino acid sequences with human C3 amino acid sequence is approx.90.7% (91%) for H5, and 96.3% (96%) for H6. Reference is made in thisregard to FIG. 2 showing various embodiments of the invention, includinghybrids H5 and H6.

In order to retain decomplementing activity, the proteins require thepresence of at least a stretch of 43 CVF amino acids in the carboxyterminal region, namely amino acids 1575 to 1617 of SEQ ID NO:3, whichreplace amino acids 1596 to 1638 of the C3 sequence (cf. FIG. 2, H7truncated). The 43 amino acid stretch may either directly form thecarboxy terminus of the hybrid protein or may be embedded within alarger sequence part comprising, for example, up to 118 CVF amino acidsas is the case for H6 (i.e. replacing amino acids 1546 to 1663 of the C3sequence), or up to 275 CVF amino acids as is the case for H5 (i.e.replacing amino acids 1389 to 1663 of the C3 sequence). In the lattercases, the 43 amino acids are embedded within a larger CVF fragmenthaving amino acid stretches of various lengths at both ends of the 43amino acid stretch. In the case of H5 and H6, the proteins additionallycomprise the 25 carboxy terminal amino acids of the CVF sequence part(ie. amino acids 1618 to 1642 of SEQ ID NO:3), resulting in a totallength of 1663 amino acids for H5 and H6. As already outlined above, inthe hybrid proteins of the invention some or all of these 25 carboxyterminal amino acids of CVF may be lacking, resulting in a polypeptideof the invention having a length of between 1638 and less than 1663amino acids.

The invention also relates to a nucleic acid encoding a protein having alength of 1638 to 1663 amino acids, which is a derivative of humancomplement component C3 (human C3), the amino acid sequence of which isshown in SEQ ID NO:2, wherein the carboxy terminal part of at least 68amino acids of human C3 is replaced by a partial sequence of Cobra VenomFactor (CVF), the amino acid sequence of which is shown in SEQ ID NQ:4,which partial sequence comprises at least the 68 carboxy terminal aminoacids of CVF or a fragment thereof lacking 1 to 25 carboxy terminalamino acids, wherein said protein has at least 70% identity to human C3or a fragment thereof, the fragment comprising at least amino acids 1 to1638 of SEQ ID NO:2.

According to a preferred embodiment, the nucleic acid is a nucleic acidencoding a protein having the amino acid sequence shown in SEQ ID NO:6.Preferably, the nucleic acid has the nucleotide sequence shown in SEQ IDNO:5.

According to another embodiment, the nucleic acid is a nucleic acidencoding a protein having the amino acid sequence shown in SEQ ID NO:8.Preferably, the nucleic acid has the nucleotide sequence shown in SEQ IDNO:7.

According to a further embodiment, the nucleic acid is a nucleic acidencoding a protein having the amino acid sequence shown in SEQ ID NO:10.Preferably, the nucleic acid has the nucleotide sequence shown in SEQ IDNO:9.

Further provided is a nucleic acid encoding a protein having the aminoacid sequence shown in SEQ ID NO:12. Preferably, the nucleic acid hasthe nucleotide sequence shown in SEQ ID NO:11.

As used herein, the term “nucleic acid molecule” refers to apolynucleotide of natural or synthetic origin, which can be single ordouble stranded, can correspond to genomic DNA, cDNA or RNA, and canrepresent either the sense or antisense strand or both.

The term “nucleic acid molecule” is intended to include nucleic acidmolecules that contain one or more non-natural nucleotides, such asnucleotides having modifications to the base, the sugar, or thephosphate portion, or having one or more non natural linkages, such asphosphothioate linkages. Such modifications can be advantageous inincreasing the stability of the nucleic acid molecule, particularly whenused in hybridization applications.

Furthermore, the term “nucleic acid molecule” is intended to includenucleic acid molecules modified to contain a detectable moiety, such asa radiolabel, a fluoro-chrome, a ferromagnetic substance, or adetectable binding agent such as biotin. Nucleic acid moleculescontaining such moieties are useful as probes for detecting the presenceor expression of C3-derivative nucleic acid molecule.

The invention is also directed to expression of the protein in suitablehost cells. In this context, a vector is provided which comprises anucleic acid, which nucleic acid encodes a protein having a length of1638 to 1663 amino acids, which is a derivative of human complementcomponent C3 (human C3), the amino acid sequence of which is shown inSEQ ID NO:2, wherein the carboxy terminal part of at least 68 aminoacids of human C3 is replaced by a partial sequence of Cobra VenomFactor (CVF), the amino acid sequence of which is shown in SEQ ID NO:4,which partial sequence comprises at least the 68 carboxy terminal aminoacids of CVF or a fragment thereof lacking 1 to 25 carboxy terminalamino acids, wherein said protein has at least 70% identity to human C3or a fragment thereof, the fragment comprising at least amino acids 1 to1638 of SEQ ID NO:2.

Preferably, the vector comprises a nucleic acid, which nucleic acidencodes a protein having an amino acid sequence, which amino acid isselected from the group consisting of:

a) the amino acid sequence shown in SEQ ID NO;6;

b) the amino acid sequence shown in SEQ ID NO:8;

c) the amino acid sequence shown in SEQ ID NO:10; and

d) the amino acid sequence shown in SEQ ID NO:12

According to a preferred embodiment, the vector comprises a nucleicacid, which nucleic acid has a nucleotide sequence selected from thegroup consisting of:

a) the nucleotide sequence shown in SEQ ID NO:5;

b) the nucleotide sequence shown in SEQ ID NO:7;

c) the nucleotide sequence shown in SEQ ID NO:9; and

d) the nucleotide sequence shown in SEQ ID NO:11.

The constructs represented by SEQ ID NO:5 and SEQ ID NO:7 arehereinafter also termed ‘H5’ and ‘H6’, respectively.

In the aforementioned expression vectors, the nucleic acid molecules areoperatively linked to a promoter of gene expression. As used herein, theterm “operatively linked” is intended to mean that the nucleic acidmolecule is positioned with respect to either the endogenous promoter,or a heterologous promoter, in such a manner that the promoter willdirect the transcription of RNA using the nucleic acid molecule as atemplate. The invention provides nucleic acid molecules which areoperatively linked to said promoter as well as vectors comprising saidpromoter driven nucleic acid molecules.

Methods for operatively linking a nucleic acid to a heterologouspromoter are well known in the art and include, for example, cloning thenucleic acid into a vector containing the desired promoter, or appendingthe promoter to a nucleic acid sequence using polymerase chain reaction(PCR). A nucleic acid molecule operatively linked to a promoter of RNAtranscription can be used to express C3-derivative transcripts andpolypeptides in a desired host cell or in vitrotranscription-translation system. The choice of promoter to operativelylink to a invention nucleic acid molecule will depend on the intendedapplication, and can be determined by those skilled in the art.Exemplary promoters suitable for mammalian cell systems include, forexample, the SV40 early promoter, the cytomegalovirus (CMV) promoter,the mouse mammary tumor virus (MMTV) steroid-inducible promoter, and theMoloney murine leukemia virus (MMLV) promoter. Exemplary promoterssuitable for bacterial cell systems include, for example, T7, T3, SP6and lac promoters.

Exemplary vectors of the invention include vectors derived from a virus,such as a bacteriophage, a baculovirus or a retrovirus, and vectorsderived from bacteria or a combination of bacterial sequences andsequences from other organisms, such as a cosmid or a plasmid. Thevectors of the invention will generally contain elements such as anorigin of replication compatible with the intended hast cells;transcription termination and RNA processing signals; one or moreselectable markers compatible with the intended host cells; and one ormore multiple cloning sites. Optionally, the vector will further containsequences encoding tag sequences, such as GST tags, and/or a proteasecleavage site, such as a Factor Xa site, which facilitate expression andpurification of the encoded polypeptide.

The choice of particular elements to include in a vector will depend onfactors such as the intended host cells; the insert size; whetherexpression of the inserted sequence is desired; the desired copy numberof the vector; the desired selection system, and the like. The factorsinvolved in ensuring compatibility between a host cell and a vector fordifferent applications are well known in the art.

Also provided are cells containing an isolated nucleic acid moleculeencoding a C3-derivative of the invention. The isolated nucleic acidmolecule will generally be contained within a vector. The cells of theinvention can be used, for example, for molecular biology applicationssuch as expansion, subcloning or modification of the isolated nucleicacid molecule. For such applications, bacterial cells, such aslaboratory strains of E. coli, are useful, and expression of the encodedpolypeptide is not required. The cells of the invention can alsoadvantageously be used to recombinantly express and isolate the encodedpolypeptide. For such applications, bacterial cells (e.g. E. coli),insect cells (e.g. Drosophila), yeast cells (e.g. S. cerevisiae), andvertebrate cells (e.g. mammalian primary cells and established celllines; and amphibian cells, such as Xenopus embryos and oocytes), can beutilized.

The polypeptides described herein, which are referred to, for example,as C3-derivatives or hybrid proteins, are therapeutic compounds that canbe administered to individuals, in particular to human subjects, fordecomplementation.

According to a preferred embodiment, the invention provides the use ofsaid polypeptides in a method for treating a patient suffering fromcomplement-associated disorders or disorders affected by complementactivation, comprising administering an effective amount of said proteinor polypeptide, respectively. Specifically, the complement-associateddisorder includes but is not limited to asthma, systemic lupuserythematodes, glomerulonephritis, rheumatoid arthritis, Alzheimer'sdisease, multiple sclerosis, myocardial ischemia, reperfusion, sepsis,hyperacute rejection, transplant rejection), cardiopulmonary bypass,myocardial infarction, angioplasty, nephritis, dermatomyositis,pemphigoid, spinal cord injury, and Parkinson's disease

The invention thus relates to the use of the polypeptides or proteins ofthe invention for preparing a pharmaceutical composition fordecomplementation, for treating a patient suffering fromcomplement-associated disorders or disorders affected by complementactivation (see above).

The invention further provides a pharmaceutical composition comprisingthe protein or polypeptide of the invention. Specifically, apharmaceutical composition of the invention comprises an effectiveamount of a protein having an amino acid sequence, said amino acidsequence being selected from the group consisting of:

a) the amino acid sequence shown in SEQ ID NO:6;

b) the amino acid sequence shown in SEQ ID NO:8;

c) the amino acid sequence shown in SEQ ID NO:10; and

d) the amino acid sequence shown in SEQ ID NO:12.

The therapeutic compounds can be administered to a mammal or human,respectively, by routes known in the art including, for example,intravenously, intramuscularly, subcutaneously, intraorbitally,intracapsularly, intraperitoneally intracisternally, intra-articularly,intracerebrally, rectally, topically, intranasally, or transdermally.Preferred routes for human administration are intravenousadministration. The pharmaceutical compositions of the invention arethus formulated for said administration routes, and, according to apreferred embodiment, comprise the compound and a pharmaceuticallyacceptable carrier depending on the route of administration of thecompound and on its particular physical and chemical characteristics.Pharmaceutically acceptable carriers are well known in the art andinclude sterile aqueous solvents such as physiologically bufferedsaline, and other solvents or vehicles such as glycols, glycerol, oilssuch as olive oil and injectable organic esters. A pharmaceuticallyacceptable carrier can further contain physiologically acceptablecompounds that stabilize the compound, increase its solubility, orincrease its absorption. Such physiologically acceptable compoundsinclude carbohydrates such as glucose, sucrose or dextrans;antioxidants, such as ascorbic acid or glutathione; chelating agents;and low molecular weight proteins.

The following examples are intended to illustrate but not limit thepresent invention.

EXAMPLE I

1 Materials and Methods

1.1 Materials

1.1.1 Chemicals

General chemicals were purchased from Sigma (Taufkirchen, Germany),Merck (Darmstadt, Germany), Fluka (Buchs, Switzerland), Invitrogen(Leek, Netherlands), Biomol Feinchemikalien (Hamburg, Germany), GibcoBRL (Eggenstein, Germany), Applichem (Darmstadt, Germany), Roth(Karlsruhe, Germany), Millipore (Eschborn, Germany), Calbiochem(Schwalbach, Germany) and Peqlab (Erlangen, Germany).

1.1.2 Enzymes, Proteins and Antibodies

Restriction enzymes, Mung Bean nuclease, T4-Ligase, Calf intestinalalkaline phosphatase (CIAP) and the respective buffers were purchasedfrom MBI Fermentas (St. Leon-Rot, Germany) and at NEB (Frankfurt,Germany). Thermus aquaticus DNA-polymerase was purchased from AGS(Heidelberg, Germany) or Roche (Mannheim, Germany). Lysozyme was alsopurchased from Roche (Mannheim, Germany). RNase was purchased from Sigma(Taufkirchen, Germany). CVF, C3 and Factor B are commercially availablefrom Calbiochem (Schwalbach, Germany).

Antisera with specificity for CVF, C3 and Factor B can be generatedfollowing standard immunization procedures. Factor D was purchased fromSigma (Taufkirchen, Germany). Strep-Tactin was purchased from IBA(Gottingen, Germany). Antiserum against C3 from goat was purchased fromCappel (Eschwege, Germany). A monoclonal antibody against C3d waspurchased from Quidel (Heidelberg, Germany). The monoclonalanti-Strep-tagII antibody was purchased from IBA (Gottingen, Germany).The secondary antibody anti-rabbit, anti-mouse or anti-goat, conjugatedwith alkaline phosphatase or peroxdase was also purchased from Sigma(Taufkirchen, Germany) or Cappel (Eschwege, Germany).

1.1.3 Affinity Matrix and Particles

Ni-NTA-agarose was purchased from Qiagen (Hilden, Germany).Strep-Tactin-Sepharose was purchased from IBA (Göttingen, Germany) andprotein A/G-agarose was purchased from Santa Cruz Biotechnology (SantaCruz, Calif., USA).

1.1.4 Bacteria and Yeast Strains

The bacteria strain E. coli DH5α (Promega, Mannheim, Germany) was usedfor amplification of plasmids. In Table 1, the genotypes of the strainsare listed.

TABLE 1 Genotypes of the bacteria strains used. DH5α F⁻, endA1, gryA96,thi-1 hsdR17 (r_(K) ⁻, m_(K) ⁺), supE44, relA1. Φ80ΔlacZΔM15, Δ(lacZYA-argF), U169

1.1.5 Vectors

The commercially available vectors which were used are listed. Thevectors which are not commercially available are depicted in FIGS. 3 and4.

pUC18

Vector pUC18 (MBI Fermentas, St. Leon-Rot, Germany) was used forcloning.

pEGFP-N1

Vector pEGFP-N1 (Clontech, Heidelberg, Germany) was used fortransfection of mammalian cell lines in order to determine thetransfection efficiency.

pcDNA3

Vector pcDNA3 (Invitrogen, Leek, Netherlands) was used for eukaryoticexpression of proteins.

pUC18CVF*/pcDNA3CVF

Plasmids pUC18CVF* and pcDNA3CVF contain the cDNA of CVF (GenebankAccession No. U09969). In pUC18CVF*, a BglII-restriction site wasintroduced in Position 1793 (Mutation A1797C) and a HindIII-restrictionsite was deleted in Position 2380 (A2380T, G2381C). Plasmid pcDNA3CVF isshown in FIG. 3.

pUC18hC3/pcDNA3hC3

The plasmids contain the cDNA of human C3 (FIG. 4).

1.1.6 Cell Lines and Culture

GMEM medium, DMEM medium, penicillin G, streptomycin, geneticin andtrypsin/EDTA were purchased from Gibco BRL (Eggenstein, Germany).Adenosine, guanosine, cytosine, uracil, thymidine, L-aspartic acid, andL-glutamic acid were purchased from Sigma-Aldrich (Steinheim, Germany).Culture flasks were purchased from Greiner (Frickenhausen, Germany),Nunc (Wiesbaden, Germany) or Sarstedt (Nümbrecht, Germany). Fetal calfserum was purchased from Biochrom (Berlin, Germany).

Cell lines CHO, COS-7 and HEK293 were used for expression. CHO-cellswere cultured in GMEM with 10% FCS and other additives. HEK293 andCOS7-cells were cultured in DMEM with 10% FCS.

1.1.7 Solutions

BCIP-stock solution 0.5% (w/v) BCIP in DMF CAPS-buffer 20 mM CAPS, pH11.0 10% methanol citrate buffer 50 mM citric acid, pH 4.0Coomassie-destainer 45% (v/v) methanol 10% (v/v) glacial acidic acidCoomassie-staining solution 0.25% (w/v) Coomassie Brillant Blue R-25045% (v/v) methanol 10% (v/v) glacial acidic acid detection buffer (forAP) 0.1 M Tris-HCl 4 mM MgCl₂ pH 9.5 detection buffer (for POD) 3.3 mgABTS 15 ml citrate buffer, pH 4.0 26.5 μl H₂O₂ developer 0.26 M sodiumcarbonate (silver staining) 0.6% (v/v) formaldehyde (37%) stainingsolution 6 mM silver nitrate (silver staining) 0.6% (v/v) formaldehyde(37%) fixing solution 30% (v/v) ethanol (silver solution) 10% (v/v)glacial acidic acid FCS 30 min heat-inactivated at 56° C., storage in 50ml aliquots at −20° C. G418-stock solution 0.4% (w/v) G418 in HEPES (100mM), pH 7.4 (40 mg/ml) filter sterile, storage at −20° C. G + A 600 mgL-Aspartamic acid 600 mg L-Glutamic acid ad 100 ml ddH₂O filter sterile,storage at 4° C. GVBS⁺⁺ 0.1% gelatine in VBS⁺⁺, dissolve at 50° C.,storage at 4° C. incubation solution 25% (v/v) ethanol (silver staining)14 mM sodium thiosulfate 0.5 M sodium acetate 0.5% (v/v)glutardialdehyde (25%) incubation buffer 0.1 M NaHCO₃, pH 9.5 loadingdye (6x) 20% (w/v) Ficoll 400 100 mM EDTA 0.025% (w/v) bromphenol-blue0.025% (w/v) xylen xyanol FF Solution I 50 mM glucose 25 mM Tris-HCl, pH8.0 10 mM EDTA autoclave, storage at 4° C. Solution II 0.2 M NaOH 1% SDSSolution III 3 M potassium acetate 11.5% acidic acid autoclave, storageat 4° C. NBT-stock solution 0.1% (w/v) NBT in 0.1 M Tris-HCl, pH 9.5nucleoside 175 mg adenosine 175 mg guanosine 175 mg cytosine 175 mguracil 60 mg thymidine ad 500 ml dH₂O filter sterile PBS (5x) 68.4 mMNaCl 13.4 mM KCl 7.3 mM KH₂PO₄ 40 mM NaH₂PO₄, pH 7.4 phenol/chloroform50% (v/v) phenol (Tris saturated) 50% (v/v) chloroform sample buffer(4x) 250 mM Tris-HCl, pH 6.8 8% (w/v) SDS 40% (v/v) glycerin 0.004%(w/v) bromphenol-blue stacking gel buffer (4x) 0.5 M Tris-HCl 0.4% SDSpH 6.8 SDS-stock solution 10% (w/v) SDS stop solution 50 mM EDTA (silverstaining) TAE-buffer (50x) 2 M Tris-acetate 20% 0.5 M EDTA pH 8.0 tankbuffer (1x) 25 mM Tris-HCl 192 mM glycin 0.1% SDS TBS (5x) 100 mM Tris250 mM sodium chloride pH 7.5 TBST 0.05% (v/v) Tween 20 in TBSseparation gel buffer (4x) 150 mM Tris-HCl 0.4% SDS pH 8.8 Tris-HCl pH9.5 100 mM Tris 4 mM MgCl₂ pH 9.5 VBS 2.5 mMsodium-5,5-diethylbarbituric acid 143 mM NaCl pH 7.4 VBS⁺⁺ 0.15 mM CaCl₂0.75 mM MgCl₂ in VBS, pH 7.4 washing buffer 0.1% (v/v) Tween 20 in PBS

1.1.8 Media

GMEM-medium 50 ml BHK21 medium 10x (Glasgow MEM) (without FCS) 18.1 mlsodium bicarbonate, 7.5% 5 ml G + A 10 ml nucleoside 5 ml sodiumpyruvate, 100 mM 5 ml NEAA (Non-essential amino acids, 100x) 450 mlddH₂O filter sterile (0.2 μm, Surfactant-free cellulose acetate-filterunits, Nunc, Wiesbaden, Germany) GMEM-medium 50 ml BHK21 medium 10x(with FCS) 18.1 ml sodium bicarbonate, 7.5% 5 ml G + A 10 ml nucleoside5 ml sodium pyruvate, 100 mM 5 ml penicillin-streptomycin-solution 5 mlNEAA (100x) 400 ml ddH₂O 50 ml FCS filter sterile (0.2 μm,Surfactant-free cellulose acetate-filter units, Nunc, Wiesbaden,Germany) LB-medium 10 g NaCl 5 g yeast-Extract 10 g bacto-trypton ad 1 1ddH₂O, autoclave LB-agar 10 g NaCl 5 g yeast-extract 10 g bacto-trypton15 g agar ad 1 1 ddH₂O, autoclave TSS-solution 85% (v/v) LB-medium 10%(w/v) PEG 8000 5% (v/v) DMSO 50 mM MgCl₂, pH 6.5 autoclave

1.1.9 Oligonucleotides

The oligonucleotides which were used were synthesized by Metabion(Martinsried, Germany) (Table 2).

TABLE 2 List of the oligonucleotides used. Name Sequence 5′-3′ AJS01GGATCCAGGTGCTCGGGTTGG AS03 AGTACCTTCCGGCTCAGCACAACCTC C S23StrepICGGAGGTACCATGGAGAGGATGGCTC TCTAT AS26 GATAGACACGTGGAAATTTTCATTGC StrepIVCG AS34StrepV GTCTTTTTCGAACTGCGGGTGGCTCC ACCCATGAGAAGACCCTGGAAAS35StrepVI ACCCGCAGTTCGAAAAAGACGATGAC GATAAAGCTCTCTACACCCTCATCAC CCCAS36StrepV TCGAACTGCGGGTGGCTCCACCCCAG hC3 AGCCAGGGGGAGG S37StrepVIhC3ACCCGCAGTTCGAAAAAGACGATGAC GATAAAAGTCCCATGTACTCTATCAT CACC S50H8forTATGTGTACAAAACCAAGCTGCTTCG AS51H8 TTCTTCTAGATTAAGTAGGGCAGCCA BackAACTCAGT AS61His5′ ATGATGATGATGATGATGCCCCAGAG hC3 CCAGGGGGAGG S62his5′CATCATCATCATCATCATGACGATGA hC3 CGATAAAAGTCCCAT

1.2 Methods

1.2.1 Densitometric Determination of Concentration

For densitometric determination of the concentration, 45 dilutions ofknown concentration of native CVF or human C3 (depending on the sample)were applied in addition to different volumina of the protein containingsample to be determined. The concentrations of the proteins of thecalibration serious was chosen so that they were located in the range ofthe proton amount which was expected in the sample. The gel wassubjected to wet-blotting or semidry-blotting procedures and theproteins were subsequently stained using immunoprinting. Then, themembrane was scanned and the concentration of the sample was determinedusing the program Imagemaster 1D Elite Version 2.01 (Amersham PharmaciaBiotech, Freiburg, Germany)

1.2.2 ELISA

ELISA was performed in order to detect proteins in supernatant. For thispurpose, 1 μg of a protein, which was diluted in 100 μl incubationsolution were immobilized on the well of an ELISA plate overnight at 4°C. Subsequently, the wells of the plate were washed three times withwashing buffer (200 μl). Then, the wells were blocked with 200 μl 5%milk powder in PBS for 5 hours at room temperature. After washing threetimes, 200 μl of the supernatant of transient expression or purifieddiluted proteins in PBS, respectively, were introduced into the wellsand incubated overnight under agitation at 4° C. After this, the sampleswere washed three times with washing buffer and incubated with 100 μl ofa 1:1000 dilution of the respective antibody in 2.5% milk powder in PBSat room temperature. Subsequently, the samples were washed three timesand incubated for one hour with 100 μl of a 1:1000 dilution of anrespective peroxidase-conjugate in a 2.5% milk powder in PBS at roomtemperature. Finally, the samples were washed three times, and then 100μl detection buffer (for POD) were added. After staining the wells,extinction at 405 nm was measured with a micro titer plate-photometer(ELISA-Reader, SLT-Instruments, Grödingen, Austria, Easy Reader EAR400AT).

1.2.3 Expression of CVF, C3 and the Hybrids in Mammalia

For expression of recombinant CVF, C3 and of the hybrids of theinvention, different cell lines ere available.

COS-7-cells were originally obtained from kidney of monkeys and containthe SV-40 origin of replication. They express the large T-antigen of theSV-40 virus, which facilitates efficient replication. As a consequence,these plasmids show high copy numbers in the cell. Thus, the cells aresuitable for transient expression. CHO-cells from the ovaria of Chinesehamster contain an RNA polymerase gene having a nucleus localisationsignal. Therefore, they can be used for preparation of stablytransfected cell lines. HEK293-cells are human embryonic kidney cellsand can be used for transient or stable expression. The HEK.EBNA-cellswhich were employed constitutively express the Epstein-Barr-Virus (EBV)Nuclear Antigen 1 (EBNA-1, EBV nuclear antigen 1). EBNA-1 was identifiedas the gene which is mainly responsible for immortalization of cells bythe EBV (Lupton and Levine, 1985). For expression, vector pcDNA3 wasavailable, which facilitates an efficient expression in mammalia via theCMV-promotor. The neomycin gene is available as resistence for theexpression under selection pressure. The expression was performed asdescribed in 1.2.9.

1.2.4 Partial Purification of the Recombinant CVF From the Supernatantof the Transient Expression

For separation of the low molecular components of the culturesupernatants or column fractions, these supernatants or fractions weretransferred into a dialysis tube (SpectraPor CE 100, MWCO 100 kDa, Roth,Karlsruhe, Germany) and dialysed overnight against PBS.

The concentration of dialysed samples was performed with Centricon-units(MWCO 100 kDa, Millipore, Eschborn, Germany). For this purpose,centrifugation was performed at 1,000 xg (4° C.) until the desired finalvolume was reached.

For partial purification and for separation of interfering components,respectively, as well as of low molecular components, columnchromatographic methods were employed. The supernatant (2.5 ml) fromtransient expression was loaded onto a PD10-column according to themanufacturer's instructions (Amesham Pharmacia Biotech, Freiburg,Germany). PBS was used as a running buffer. The fractions were examinedfor recombinant protein by Western blotting and subsequentimmunoprinting. For partial purification, 2 ml supernatant of thetransient expression wee loaded onto a 1 ml EconoPac-column (Biorad,Munich, Germany) using a Trisbuffer (50 mM, pH 7.5). The same bufferwith 500 mM NaCl added thereto was used for elution. The fractions werealso examined for protein content and dialysed against PBS.

For control purposes, the identic procedure was employed with thesupernatant of non-transfected cells.

1.2.5 Purification and Detection of Proteins Using the Strep-tag System

1.2.5.1 The Strep-tag System

For purification and immobilization of the recombinant proteins, CVF, C3and the hybrids consisting of CVF and C3 were supplied with aStrep-tag-fusion peptide. The Strep-tag is a synthetic peptideconsisting of 9 amino acids (AWRHPQFGG), which binds to streptavidinwith an affinity of 2.7×10 ⁴ (Schmidt et al., 1996). It uses the bindingpocket for biotin. As C-terminal fusion partner for proteins, it can beused for purification and detection. An N-terminal fusion is alsopossible since the system has been improved. The resulting Strep-tagII(WSHPQFEK) binds with a lower affinity to Streptavidin (Schmidt et al.,1996), however, a derivate was found by a selection round with randomlymutated Streptavidin, which in turn has a sufficient high affinity(Skerra and Schmidt, 2000). With a dissociation constant of 1 μM, theagarose-immobilized Streptavidin-derivate Strep-Tactin can be used forpurification. Strep-tactin-conjugates or anti-Strep-tagII antibodies canbe utilized for detection in immunoprinting or in ELISA analysis.

1.2.5.2 Detection of Strep-tag Fusion Proteins

For detection of strep-tagII-fusion proteins in supernatant of transientexpression, an ELISA was conducted. For this purpose, 3 μg strep-tactin(diluted in 40 μl incubation solution) were immobilized. overnight at 4°C. on the surface on the wells of an ELISA plate (Greiner,Frickenhausen, Germany). Subsequently, the procedure described above inconnection with the ELISA was performed.

1.2.6 Purification of Hydrid H5

Supernatant (500 ml) obtained from stably transfected cells was adjustedto pH 7.5, passed trough a 0.45 μm cellulose acetate membrane and loadedonto a Poros HQ/M anion exchange column equilibrated with 50 mM Tris, pH7.5 using ÄKTA purifier (Amersham Bioscience, Freiburg, Germany). Therecombinant protein was eluted using a linear (0-500 mM) NaCl gradient.Fractions (2 ml) were analyzed using 7.5% SDS-PAGE and western blotting,pooled and dialyzed against phosphate buffered saline (PBS). The pooledsample was diluted (1:9) in 50 mM sodium phosphate, 0.55 M sodiumsulfate buffer, pH 7.0, filtered (0.2 μm) and applied to a thiophilicresin (1.5 ml, BD Bioscience, Heidelberg, Germany) equilibrated with 50mM sodium phosphate, 0.5 M sodium sulfate buffer, pH 7.0. Afterextensive washing of non-adsorbed proteins with the equilibration buffer(>30 column volumes), elution was performed using 50 mM sodium phosphatebuffer, pH 7.0. Fractions (1.5 ml) were analyzed by 7.5% SDS-PAGE andwestern blotting. Fractions containing H5 were pooled, dialyzed against100 mM Tris, 150 mM NaCl, pH 8.0 (bufferW), loaded onto Strep-Tactinsepharose (2 ml, IBA, Göttingen, Germany) equilibrated with buffer W,washed with 10 ml buffer W, and eluted with buffer W containing 2.5 mMdesthiobiotin. Protein concentration and purity of the fractions wereanalyzed by 7.5% SDS-PAGE. Pooled fractions were dialyzed against PBSand employed for further characterization.

1.2.7 Purification of Hybrid His-H6

For purification of His-H6 using IMAC, imidazol was added to 50 mlstable supernatant and incubated over night on a shaking unit. Thematrix was then centrifuged (700 xg, 10 min, 4° C.), the supernatantremoved, and the matrix was resuspended in 50 ml PBS. Followingcentrifugation (700 xg, 10 min, 4° C.), the matrix was again resuspendedin 5 ml PBS and loaded onto a flowthrough column. Subsequently, boundproteins were eluted from the column in 1 ml portions using 3 ml 300 mMimidazole in PBS. Then, protein content was determined in the fractionsby SDSPAGE followed by subsequent Western blot and immunoprinting.Suitable fractions were combined, dialysed against PBS and used forfurther analysis.

1.2.8 Complement Methods

1.2.8.1 Preparation of Sensitized Sheep Erythrocytes

Sheep whole blood (1 ml, Behringwerke, Marburg) was resuspended in 13 mlcold GVBS⁺⁺ and centrifuged (10 min, 1,000 xg, 4° C.). Supernatant wasremoved, and the erythrocytes were again resuspended in 14 ml GVBS⁺⁺.This procedure was repeated until supernatant became clear.Subsequently, the erythrocytes were resuspenden in approx. 5 ml GVBS⁺⁺.Erythrocytes were adjusted by diluting with GVBS⁺⁺ to give an absorptionof 1.9 (5×10⁸ cells(ml) at 412 nm for 30 μl erythrocyte suspension in 1ml ddH₂O. 2 μl antiserum against sheep erythrocytes (anti sheep redblood cell stroma, Sigma, Taufkirchen) were added to each of 1 ml of theadjusted erythrocytes. Sensitizing was performed for 1 h in a water bathat 37° C., while inverting the reaction tube regularly after 10 min. Thesensitized erythrocytes were washed 3 times with 2 ml GVBS⁺⁺ andcentrifuged (3 min, 1,000 xg, 4° C.). The erythrocytes could be storedup to three days. Prior to each use, OD₁₂ was adjusted to 1.9.

1.2.8.2 Isolation of Guinea Pig-Erythrocytes

Guinea pig-erythrocytes were isolated from whole blood, which wasobtained from isofluoran-narcotized guinea pigs by punction of the eyes,Approximately 1 ml blood was taken and immediately transferred into atube containing 1 ml ice cold ACD solution. The ACD solution serves asanti-coagulant. The erythrocytes were separated by centrifugation (1,000xg, 4 min, 4° C.), resuspended in 14 ml GVBS⁺⁺ and again centrifuged(1,000 xg, 4 min., 4° C.). This procedure was repeated 3 to 4 timesuntil the supernatant remained clear. Finally, the erythrocytes wereresuspended in approximately 5 ml GVBS⁺⁺. The erythrocytes were dilutedwith GVBS⁺⁺ until 30 μl of the erythrocyte suspension in 1 ml ddH₂O gavean absorption of 1.9 (5×10⁸ cells/ml) at 412 nm.

1.2.8.3 Complement Consumption Assay

This test is based on the complement consuming effect of CVF. If a CVFcontaining sample is incubated with human serum, the complement proteinsare consumed depending on the CVF activity (Ballow and Cochrane, 1969;Cochrane et. al., 1970). The remaining complement activity of the serumcan be detected subsequently using sensitized sheep erythrocytes.

First, quantification of human serum was performed by serum titration,which led to hemolysis of sheep erythrocytes by 70-90%. For thispurpose, different serum concentrations (serum value) were provided in 2ml reaction tubes (double measurements) and filled up to 40 μl withGVBS⁺⁺. Additionally, controls were prepared, which contained 40 μlGVBS⁺⁺ (buffer-control) only, or 40 μl ddH₂O (complete lysis) only,respectively. All reactions were incubated for 30 min at 37° C. underagitation (Thermomixer 5437, Eppendorf, Hamburg, Germany). Then, 100 μlcold GVBS⁺⁺ or 100 μl ddH₂O (upon complete lysis), respectively, and 30μl sensitized sheep erythrocytes were added. Further incubation tookplace for 30 min, as described above. Subsequently, the samples werekept on ice. 850 μl cold VBS⁺⁺ or 850 μl ddH₂O (upon complete lysis),were added, respectively. The supernatants were transferred intocuvettes, and optical density was measured at 412 nm. Hemolysis wassubsequently calculated according to the following formula:

${\%\mspace{14mu}{hemolysis}} = {\frac{{{OD}_{412}\mspace{14mu}{serum}\mspace{14mu}{value}} - {{OD}_{412}\mspace{14mu}{buffer}\mspace{14mu}{control}}}{{{OD}_{412}\mspace{14mu}{complete}\mspace{14mu}{lysis}} - {{OD}_{412}\mspace{14mu}{buffer}\mspace{14mu}{control}}} \times 100\%}$

For complement consumption assay, the amount of serum determined inprior tests and the samples (max. 20 μl) were provided in a 2 mlreaction tube (double measurements) and supplemented with GVBS⁺⁺ to give40 μl. Additionally, reaction mixes of the following controls wereprepared: determined amount of serum, supplemented with GVBS⁺⁺ to give40 μl (serum control, 4 to 5 samples); 40 μl GVBS⁺⁺ (buffer control) and40 μl ddH₂O (complete lysis). All reaction mixes were incubated for 3hours at 37° C. under agitation. Then, 100 μl cold GVBS⁺⁺ or 100 μlddH₂O upon complete lysis, respectively, and 30 μl adjusted sensitizedsheep erythrocytes were added, and incubation took place for 30-40 min,as described above. After 15 min, serum control as well as a reactionmix of the complete lysis were measured according to the followingprinciple. The sample were kept on ice, 850 μl cold VBS⁺⁺ or 850 μlddH₂O at complete lysis, respectively, were added and centrifuged (4°C., 2,000 xg, 2 min.). Supernatants were transferred into cuvettes, andthe optical density was measured at 412 nm. Lysis was calculatedaccording to the following formula:

${\%\mspace{14mu}{hemolysis}} = {\frac{{{OD}_{412}\mspace{14mu}{serum}\mspace{14mu}{control}} - {{OD}_{412}\mspace{14mu}{buffer}\mspace{14mu}{control}}}{{{OD}_{412}\mspace{14mu}{complete}\mspace{14mu}{lysis}} - {{OD}_{412}\mspace{14mu}{buffer}\mspace{14mu}{control}}} \times 100\%}$

In case the value for the serum control was clearly below 80% of thecomplete lysis value, a second serum control was taken after 10 furtherminutes of incubation and measured as described above. Once a value of70-80% hemolysis was obtained, all reaction mixes were measured andevaluated according to the same principle In order to facilitatecomparison of different series of measurements, values were referred tothe corresponding serum control.

1.2.8.4 Solid Phase Complement Consumption Assay

For characterization of complement consuming activity of the recombinantstrep-tagII-C3/CVF hybrids, a solid phase assay was performed. Here,proteins were bound via their strep-tag to an ELISA plate, which wascovered with strep-tactin. Subsequently, activity can be determined byadding serum. For this purpose, 3 μg strep-tactin (diluted in 40 μlincubation solution) were immobilized overnight at 4° C. on the surfaceof the wells of an ELISA plate (Greiner, Frickenhausen, Germany). Then,the wells were washed 3 times with 200 μl washing buffer andsubsequently blocked with 200 μl 3% BSA in PBS for 5 hours at roomtemperature. After washing the wells again (3 times), different volumesof the supernatants or of the samples were provided in the wells.Protein concentration in the supernatants was previously determineddensitometrically. After over night incubation at 4° C. under agitation,the wells were washed 3 times with washing buffer. Subsequently, GVBS⁺⁺and the amounts of serum determined in serum titration were added,giving a volume of 60 μl. The ELISA plate was sealed with paraffin andfixed to the bottom of an incubation shaker. Subsequently, incubationtook place for 3 hours at 37° C., 150 rpm. Supernatants were transferredinto 2 ml reaction tubes, and following addition of 100 μl GVBS⁺⁺ and 30μl sensitized erythrocytes the complement consumption assay wasconducted as described.

1.2.8.5 Bystander Lysis-assay

This test for detecting hemolytic activity is based on fluidC5-activation and can be determined by lysis of non-sensitized guineapig erythrocytes (Vogel, 1985). In this method, the extent of complementactivation is determined by photometric measurement of releasedhemoglobin. Different amounts of the protein to be analyzed were dilutedin 20 μl GVBS++ mixed with 20 μl guinea pig serum (Sigma, Taufkirchen,Germany) and 20 μl guinea pig erythrocytes (5×108 cells/ml) in a 2 mlreaction tube and incubated for 3 hours at 37° C. under agitation(Thermomixer 5436, Eppendorf, Hamburg, Germany). The reaction wasstopped by adding 1 ml ice cold VBS++-buffer. The erythrocytes werecentrifuged (2,000 xg, 4° C., 2 min) and released hemoglobin in thesupernatant was determined by measurement of extinction at 412 nm.Reaction mixes with 20 μl erythrocytes and 40 μl ddH2O (complete lysis)or 20 μl guinea pig serum, 20 μl erythrocytes and 20 μl GVBS++ (serumcontrol), respectively, served as controls.

1.2.8.6 Determination of the Stability of C3 Convertases

500 ng of each native CVF (nCVF), hC3, and the derivative H5 were mixedwith 950 ng of factor B and 8 ng of factor D in a volume of 60 μl VBS(2.5 mM Na-5,5-diethyl-barbituric acid, 143 mM NaCl, pH 7.4). Afteraddition of MgCl₂ to a final concentration of 10 mM, the samples wereincubated for 2 h at 37° C. to allow for convertase formation.Subsequently, all samples were supplemented with EDTA to a finalconcentration of 10 mM to inhibit further formation of convertases.Thereafter, the samples were incubated at 37° C. and after differentperiods of incubation 20 μl aliquots were added to 150 μMBoc-Leu-Gly-Arg-7-amido-4-methylcoumarin acetate (Sigma, Taufkirchen,Germany) in 180 μl VBS. The timecourse of fluorophore release wasdetermined in black FIA-Plates (96 K, Greiner Bio-One, Frickenhausen,Germany) using an excitation filter of 370 nm and an emission filter of465 nm in amicroplate reader (Genios, Tecan, Creilsheim, Germany).Values after 60 min of fluorophore release were defined as 100%. Theslope of the graph was used to determine the enzymatic activity of thesample.

1.2.9 Tissue Culture and Expression Methods

1.2.9.1 Culturing and Passaging of COS7 and HEK293 Cells

COS-7 cells or HEK293 cells were cultured in an incubator (HeraeusInstruments Begasungsbrutschrank 6060) in a water saturated atmosphere(5% CO₂) at 37° C. Growth was performed in DMEM medium (Gibco/BRL,Eggenstein, Germany) which was supplemented with 10% FCS (Biochrom,Berlin, Germany). As soon as cells grew confluently in the tissueculture flasks (75 cm³, Cellstar, Greiner Labortechnik, Frickenhausen,Germany), they were passaged into a new culture flask (approximatelyevery 3 days). Cell supernatant was removed and the cells were washedwith PBS. 4 μl trpysin/EDTA (Gibco BRL, Eggenstein, Germany) were added,and the cells were incubated for 5 min in an incubator. Detachment ofthe cells from the bottom was supported by gentile knocking and wascontrolled under the microscope. When the cells were almost completelydetached, the procedure was stopped by adding 8 ml serum-containingmedium. The suspension was transferred into a 15 ml-reaction tube, andthe cells were sedimented by centrifugation (5 min, 1,000 xg, RT). Thesupernatant was removed, the pellet was resuspended in 10 mlserum-containing medium and 1 to 3 ml of the cell suspension weretransferred into a new tissue culture flask and supplemented with serumcontaining medium to give a final volume of 13 ml.

1.2.9.2 Culturing and Passaging CHO-Cells

Growth of cells was performed in serum-containing GMEM-medium (10% FCS).Culturing and passaging of CHO-cells was performed in an analogousmanner as described for COS-7-cells.

1.2.9.3 Transfection

For expression in mammalian systems, expression vector pcDNA3(Invitrogen, Leek, the Netherlands) comprising the corresponding geneswas introduced into the cells by the GenePorter reagent (PeqLab,Erlangen),

DNA (1 to 4 μg) and 10 to 15 μl GenePorter transfection reagent werediluted in 500 μl serum-free medium and pooled. The reaction mix wasincubated for 45 min at room temperature. At the same time, the cellswhich were passaged in a 6 well plate approx. 24 hours earlier (300-500μl of cell suspension with 2 ml serum containing medium per well;TG-plate, 6-well, Greiner Labortechnik, Frickenhausen, Germany), werewashed with PBS. Then, the DNA GenePorter mix was carefully addeddrop-wise to the cells, and the 6-well plate was placed in theincubator, After 3 hours, the medium was replaced against 2 mlserum-containing medium, and the cells were kept for 2 to 3 days in theincubator for cell growth.

When using Nutridoma HU (100; Roche Diagnostics, Mannheim, Germany), thecells were added to Nutridoma HU after transfection in serum-free mediumand kept growing in the incubator for 2 to 3 days.

A reaction mix without DNA was prepared as negative control. Ifapplicable, a reaction mix with 1 μg pEGFP-N1 was prepared as positivecontrol. Plasmid pEGFP-N1 encodes the green fluorescence protein (GFP)and can be used for determining transfection efficiency since the GFPexpressing cells can be detected using a fluorescence microscope. Thesupernatant of the pEGFPN1 transfected cells was already removed after24 hours, cells were washed with 2 ml PBS, and 500 μl trypsin/EDTA wereadded. Detachment of cells was perfumed for 5 min in the incubator.Then, 2 ml PBS were added and the cell suspension was transferred into a50 ml reaction tube for centrifugation (5 min., 1,000 xg, RT). Thesupernatant was removed and the cell pellet was resuspended in 2 ml PBS.10 μl thereof were provided in a Neubauer-counting chamber, and thenumber of fluorescening cells and the total number of all cells werecounted in the outer four squares.

Calculation of the Cell Number/ml:

${{Cells}\text{/}{ml}} = {\frac{{Number}\mspace{14mu}{of}\mspace{14mu}{cells}\mspace{14mu}{of}\mspace{14mu}{all}\mspace{14mu}{squares}}{4} \times 10^{4}}$Calculation of Transfection Efficiency:

${{Efficiency}\mspace{14mu}(\%)} = {\frac{{number}\mspace{14mu}{of}\mspace{14mu}{fluorescent}\mspace{14mu}{cells}}{{number}\mspace{14mu}{of}\mspace{14mu}{cells}\mspace{14mu}{of}\mspace{14mu}{all}\mspace{14mu}{squares}} \times 100\%}$

1.2.9.4 Expression Under Selection Pressure

In order to increase yields, generation of stably expressing lines wasdesired, wherein the reaction mixes from transient expression were keptin culture by adding an antibiotic and by sub culturing. Depending onresistance, 10 μl/ml culture medium were added to an antibiotic stocksolution (G418), or 5 μl/ml culture medium were added to zeocine.

1.2.9.5 Expression in Serum or Protein Free Medium

For culturing in serum-free or protein-free medium, cells were step-wiseadapted with SCF30- or Mampf3-medium (Promocell, Heidelberg, Germany)with 1 mM L-glutamine. The percentual portion of the serum-free orprotein-free medium was increased by 25% in every second passage.Otherwise, passaging took place as described.

1.2.9.6 Monoclonalization

In order to obtain a homogeneous cell population, monoclonalization wasconducted, wherein the different cells were monoclonalized, expanded andsubsequently examined with respect to their expression level by Westernblotting and immuno printing.

For this purpose, all cells were incubated for 3 to 4 passages aftertransfection under selection pressure in order to guarantee that a largeportion of cells contained the resistance and therefore expressed thetarget protein. This procedure allows to equate the cell number per ml,which is counted thereafter using a Neubauer counting-chamber, with thenumber of cells, which express the target protein. Subsequently, celldensity was set to 1 cell per 100 μl medium with 10% FCS, and 100 μl ofthis dilution were introduced into each of the 48-96 wells of the 96well plates (Greiner Labortechnik, Frickenhausen, Germany). Afterapproximately one week, the medium was removed, and 100 μl medium with10% FCS were added. After approximately 2 weeks, the wells were examinedunder the microscope for one colony per well. Some were selected, andthe cells were detached with 25 μl trypsine for 5 min at 37° C. andcompletely transferred into the well of a 24-well-plate (GreinerLabortechnik, Frickenhausen, Germany), which was completely filled with500 μl medium containing 10% FCS. After one week, this precedure wasrepeated with 100 μl trypsine, and all cells were placed into a well ofa 6-wellplate. After another 3 to 4 days, 100 μl supernatant per wellwere taken and examined by Western blotting and subsequent immunoprinting. The cell population, which finally showed the strongest band,was further expressed under selection pressure and cryoconserved, ifapplicable.

EXAMPLE II

Concept and Establishment of a Solid Phase Assay (cf. Example I,1.2.8.4)

The assay comprises immobilizing the hybrids as Strep-tagII fusionproteins to Strep-Tactin, which is bound to the surface of ELISA plates.The subsequent addition of buffer and serum should facilitate theconduction of the complement-consumption-assays in the ELISA plate.

For immobilization, a Strep-tagII was selected, which is a peptideconsisting of 8 amino acids (WSHPQFEK). With a dissociation constant of1 μM to Strep-Tactin, the Strep-tag is suitable for directedimmobilization as well as for protein purification and detectionpurposes.

Fusion of an Affinity Tag to CVF and C3

The presence of the affinity tag is essential for the performance of thedeveloped solid-phase-assay. The proteins which shall be analyzed need afusion tag; therefore, an enterdoinase-cleavage site was inserted intothe cDNA of CVF and C3 between the signal sequence and the N-terminus ofthe Strep-tagII which allows the cleavage of the affinity tags lateron.

Using the CVF-cDNA two amplification products were generated in two PCRreactions using the oligonucleotides S35 and AS26 and theoligonucleotides S23 and AS34. The amplification products werehybridized using PCR. The amplification product was digested using therestriction enzymes KpnI and Eco72I and ligated into a pcDNA3CVF-vectorwhich was digested analogously. The cDNA of human C3 was treated in ananalogous manner. Using the oligonucleotides S01 and AS36 and S37 andAS03, amplification products were generated and hybridized. Forinsertion, the restriction sites NotI and Bpull02I were used.

Alternatively, His tags were used as affinity tags, which—in analogy tothe Strep-tags—were inserted between the signal sequence and theN-terminus.

Briefly, for H6, two amplification products were generated using St-hC3with the oligonucleotides S01 and AS61 and with the oligonucleotides S62and AS03. The amplification products were hybridized by PCR. Then, theamplification product was inserted via the restriction sites NotI andBpull02I in an analogously digested vector. The successful transientexpression of the hybrid HisH6 was verified in a sandwich-ELISA and inan immunoblot. The densitometric quantification which was performed onthe basis of the immunoblot resulted in yields of 1-2 mg/l.

Subsequently, St-CVF, St-C3 or the hybrids His-H6 or St-H6 hybrids,respectively, were successfully expressed in CHO-cells. A densitometricquantification was performed on the basis of an immunoblot and resultedin yields, which are comparable to the respective yields of wildtypeproteins.

Evaluation of Assay Conditions

For establishing this solid-phase-assay which should offer thepossibility of directly characterizing proteins after transientexpression, an evaluation of the conditions was performed. The basis forevaluation is the adaption of the complement consumption assay to asolid phase-format. In order to determine the most suitable conditions,native CVF was used in a complement consumption assay in an ELISA plateunder variation of different parameters.

For incubating the samples in the ELISA plate agitation at 37° C., acommon incubation shaker was used. For optimizing the conditions, thereaction mixtures were measured at different rotation velocities forensuring sufficient admixture of the samples, which is necessary for theassay. Further reaction mixtures were analyzed with differentpre-incubation periods for obtaining representative values for theexpected protein concentration range of 20-200 ng protein.

The best results were obtained at a rotation velocity of 150 rpm and apre-incubation time of 3 hours, since the samples with different proteinconcentrations represent significant distinguishable values.

Activity Studies of the Strep-tag-Fusion Proteins

A prerequesite for the solid phase assay is the accessibility of theStrep-tag-fusion protein, which was examined in an ELISA analysis. Thefusion proteins St-CVF and St-C3 were selectively immobilized toStrep-Tactin on the surface on an ELISA plate. Detection of the targetproteins was performed using polyclonal sera. Both proteins could beimmobilized and detected, thus demonstrating the accessibility of theStrep-tag.

The concentration of the two proteins in the supernatant of thetransient expression was quantified densitometrically on the basis ofimmunoblot. Polyclonal sera against C3 and CVF were used. The quantifiedrecombinant proteins were then immobilized in comparable concentrationsto Strep-Tactin and utilized in a complement consumption assay underevaluated conditions.

The activity of the recombinant CVF expressed in mammalia could bedemonstrated through significant reduction of hemolysis compared tohuman C3.

The positive results which were achieved also confirm that theestablished solid phase complement consumption assay is an effectivemethod for characterizing hybrids consisting of CVF and human C3.

Discussion

The solid phase-assay enables the binding of CVF and of the hybrids viaan affinity tag to a protein immobilized on a suitable surface. In thismanner, the interfering components could be separated and a subsequentcomplement consumption assay with the immobilized proteins enables thecharacterization of the hybrids.

The Strep-tagII was introduced via oligonucleotides between the signalsequence and the N-terminus of the CVF and C3, respectively.Additionally, an enterokinase-restriction site was inserted which allowsthe cleavage of the Strep-tagII lateron.

All fusion proteins were successfully expressed in CHO-cells, and theaccessibility of the fusion peptide was confirmed by ELISA analysis. Thefusion of the Strep-tag did neither influence secretion nor expressionyield, as shown by densitometric quantification.

The performing of the complement-consumption-assay in asolid-phase-system requires an adaption of the assay conditions. Forthis reason, an evaluation of different parameters was performed.Different concentrations of nCVF were subjected to complementconsumption-assays under different conditions in an ELISA plate. Boththe rotation velocity as well as the time period of the pre-incubationwas varied. Subsequent studies under optimized conditions (150 rpm, 3 hpre-incubation with St-CVF) confirmed the general feasability of thisassay system. A significant complement-consuming activity wasdemonstrated for St-CVF. In contrast, the control protein St-C3 did notshow an activity.

The establishing of solid-phase-assays therefore allows for the firsttime the efficient characterization of transiently expressed proteinscomprising a Strep-tag-fusion peptide. The successful characterizationof the recombinant CVF confirmed that a further processing of thetwo-chain CVF is not required for activity.

EXAMPLE III

Generation and Expression of H5

Cloning and Expression of Construct H5

A hybrid was constructed, which contains the human β-chain andadditionally the humanized Factor B- and Factor H-binding sites as wellas the cleavage sites for Protease Factor I. In order to generate theconstruct, BglII restriction sites were used. Construct H5 is shown inFIG. 5. In addition to the α-chain, the γ-chain as well as the C3a andthe C3d regions were humanized.

For cloning of construct H5, a fragment consisting of pUC18 and the3′terminus of CVF was obtained from pUC18CVP* utilizing Ecl136I andBglII restriction. Subsequently, said fragment was ligated with afragment obtained from pcDNA3hC3 by EcoRI restriction, followed by mungbean nuclease digestion and BglII restriction. The latter fragment had asize of 1870 bp contained the 5′ terminus of C3 cDNA. The vector ligatedin this way (referred to as H2Δ2307 bp) contained 1800 bp of the C3 5′terminus and 1000 bp of the CVF 3′ terminus.

In order to complete construct H5, vector H2Δ2307 bp was digested withBglII. The middle region of the C3cDNA was isolated from plasmidpUC18hC3 via its BglII restriction sites and inserted into the vector.The resulting construct H5 was then inserted into an analogouslydigested pcDNA3-vector via the EagI-restriction sites (FIGS. 6; A+B).Subsequently, a Strep-tag was inserted between the signal sequence andthe N-terminus.

Expression in CHO-cells was confirmed in an ELISA via Strep-Tactin andin an immunoblot. Quantification which was performed on the basis of animmunoblot resulted in yields of 12 mg/l, wherein the polyclonal serumwas employed against C3. Since hybrid H5 has 90% identity compared tohuman C3, it can be presumed that the polyclonal serum detects bothproteins with a variance that is lower than the one of densitometricquantification.

Determination of the densitometric concentration was confirmed by asandwich ELISA. Here, a monoclonal C3d-antibody and the antibody.fragment C3-1, respectively, were immobilized on the surface of an ELISAplate and then incubated with the recombinant proteins. The detectionwas performed using a polyclonal C3-antiserum. In addition to thesamples, various concentrations of human C3 were employed. Theevaluation of the ELISA analysis confirmed the concentrations obtainedfrom the densitometric quantification.

Characterization of Hybrid H5

Following successful transient expression, hybrid H5 was used in a solidphase-assay, where CVF and human C3 served as controls (FIG. 7).

Hybrid H5 clearly showed complement-consuming activity which iscomparable to CVF. Despite of ist degree of 90% humanization, hybrid H5completely retains complement-consuming activity.

To analyze the functional activity of derivative H5 more in detail, weestablished a stably transfected CHO cell line and purified the proteinusing thiophilic and strep-tactin resins (for details see Example I,1.2.6). A detailed analysis of the complement-consuming activity of H5over a broad concentration range confirmed the CVF-like activityobserved in analyses of transiently expressed H5. As evident from FIG.8, H5 exhibits approx. 85% of the complement-consuming activity ofpurified native CVF.

Furthermore, we analyzed the formation and stability of the convertasegenerated by the derivative H5 (for details see Example I, 1.2.8.6). Asshown in FIG. 9A, the recombinant protein activates factor B byproducing Bb and Ba in the presence of factor D and Mg²⁺ in an identicalmanner as C3 (H2O) and CVF. The time dependent reduction in the releaseof 7-amido-4-methylcoumarin from a fluorogenic substrate analogue by theaction of the convertase (FIG. 9B-D) revealed a half-life of theH5-dependent convertase of approx. 5-6 hours (FIG. 9E), which is closeto the reported 7 hour half-life of the CVF-dependent convertase (Vogelund Müller-Eberhard, 1982). In contrast, the C3bBb convertase complexexhibited no activity, thereby confirming the extremely short half-lifein the range of 1 to 2 minutes (Medicus et al., 1976).

These results confirm the fact that, in addition to the CVF-α-chain,also the CVF-γ-chain as well as the C3a- and C3d-homologous regions canbe substituted against human C3 without loss of complement-consumingactivity.

EXAMPLE IV

Expression and Characterisation of Hybrid H6

Cloning and Expression of Hybrid H6

Due to the fact that the complement-consuming activity of the hybrid H5was successfully detected, a further minimizing of the CVF region wasenvisaged. If the C-termini of CVF and human C3 are compared, it isobvious that in the depicted region upstream of the Bsp1407I-restrictionsites the identify between human C3 and CVF is 56%, whereas theC-termini of the two proteins merely exhibits in an identity of 44%(FIG. 10).

For the construction of the new hybrid H6, the Bsp1407I-restriction sitewas used. The C-terminal region of CVF was selected, whereas the regionupstream of the Bsp1407I-restriction site was humanized. Hybrid H6 has96.3% identity to human C3 (FIG. 11).

For the cloning of construct H6 a 350 bp-fragment from pUC18CVF* wasamplified by PCR using the oligonucleotides S50 and AS51. This fragmentcontains the 3′-terminus of CVF. The amplification product and thevector pcDNASt-hC3 were digested with Bsp1407I and XbaI and ligated(FIG. 12).

Expression of hybrid H6 was successfully confirmed in an ELISA viaStrep-Tactin and in an immunoblot.

The concentration of hybrid H6 in the supernatant of the transientexpression was quantified densitometrically on the basis of animmunoblot, wherein polyclonal serum against C3 was employed. Thisyielded concentrations of approx. 1-2 mg/l supernatant.

Characterization of Hybrid H6

In addition to densitometric determination, a sandwich ELISA wasperformed for quantifying recombinant C3 and hybrid H6. Thequantification resulted in identical concentrations. Then, hybrid H6 wasexamined in a solid phase-assay for complement-consuming activity (FIG.13).

The activity of hybrid H6 was shown in the solid phase-assay.Humanization of the CVF-molecule was successfully performed, whereby thecomplement-consuming activity was retained. H6 was purified for furthercharacterization.

Cloning and Expression of Hybrid His-H6

For a detailed characterization of hybrid H6, purification was performedaccording to established techniques using affinity-chromatographicalmethods on the basis of the Histag-system. For this purpose, theinsertion of the His-tag was performed in analogy to the insertion ofStrep-tag between signal sequence and N-terminus.

Briefly, two amplification products were generated using St-hC3 with theoligonucleotides S01 and AS61 and with the oligonucleotides S62 andAS03. The amplification products were hybridized by PCR. Then, theamplification product was inserted via the restriction sites NotI andBpm1102I in an analogously digested vector. The successful transientexpression of the hybrid His-H6 was verified in a sandwich-ELISA and inan immunoblot. The densitometric quantification which was performed onthe basis of the immunoblot resulted in yields of 1-2 mg/l.

Purification and Characterisation of Hybrid H6

For the purification of the hybrid, stable expressing CHO-cells weremonoclonalized and expanded. Subsequently, imidazole was added to thesupernatant of the CHO-cells in a final concentration of 20 mM and themixture was incubated with Ni-NTA-matrix. The elution fractions werethen analyzed in an immunoblot. The fractions were pooled, dialyzed andquantified in a Sandwich-ELISA using a monoclonal antibody.Densitometric determination was performs on the basis of an immunoblotand it confirmed the quantification achieved with the ELISA. Proteinconcentrations of 34 μg/ml were obtained. For determining purity, thesamples were separated by SDS-PAGE, and the gel was subjected to silverstaining. It was determined that the purification procedure via theHis-tag resulted in a restricted purification, since a part of theprotein did not bind to the matrix. Furthermore, the fractions containeda strong protein background. For subsequent studies, however sufficientconcentrations of the hybrids were obtained.

For further characterization of hybrid H6, first the C3-convertaseactivity was determined in a complement consumption assay in solution(FIG. 14), where the activity of hybrid H6 can be correlated to theactivity of CVF. The activities of the proteins were reflected by asignificant reduction in hemolysis. The evaluation resulted in a 68%complement-consuming activity in compared to the activity of CVF (FIG.15).

In a Bystander Lysis-test it was determined whether hybrid H6 also hasthe ability (compared to CVF) to activate the complement system via aC5-convertase-activity in fluid phase. The guinea pig serum employed isactivated by CVF and the guinea pig erythrocytes are lysed by subsequentformation of the membrane attack complex. Subsequently, the releasedhemoglobin can be measured in the supernatant at 412 nm. It wasdemonstrated that hybrid H6 does not exert significant fluid phaseC5-convertase activity.

Discussion

Recombinant CVF was detected in the supernatant of all three mammaliancell lines (CHO, COS-7 and HEK293). The densitometrically quantifiedyields were approx. 0.5-3 mg/l.

The recombinant CVF with an apparent molecular weight of 210 kDa had thestructure of two chains. Here, the CVF-α-chain as in the native proteinis a singular chain, whereas the γ-and the β-chain are expressedtogether with regions which are homologous to the C3-fragments C3d andC3a as one chain. The processing is analogous to the processing of thetwo-chain human C3; only 4 arginine residues are removed in the twoproteins. Further processing of CVF in the cobra by a venom protease(O'Keefe et al., 1988) does not occur in mammalian and insect cells.However, the recombinant two-chain CVF from insect cells exhibits acomparable activity to native CVF (Kock, 1996).

The modification of CVF by the generation and characterization ofdifferent hybrids from CVF and human C3 led to the expression ofhybrids, the majority of which consists of human C3. In order to analyzein how far the expression systems are also suitable for such hybrids,human C3 was expressed in addition to CVF in CH-O, HEK293-andCOS-7-cells. The recombinant protein was also detected in the cellsupernatant. Recombinant C3 showed an apparent molecular weight of 210kDa and expression yields ranged from 13 mg/l, which is in compliancewith literature values (Fecke et al., 1998). For the secretoryexpression of the proteins, the native signal sequences of CVF and humanC3 were used.

Due to the fact that CHO-cells allow for stable integration and furtherexhibit higher yields compared to HEK293-cells, CHO-cells were selectedfor further studies.

Successful expression of CVF and human C3 in mammalian cells can beconsidered as a useful basis for further expression of hybrids of CVFand human C3.

Following successful expression of CVF and human C3, it was possible toestablish mammalian cells as expression systems for these hybrids.Subsequently, a method had to be developed for analyzing thecomplement-consuming activity of the hybrids. For this purpose,recombinant CVF in the supernatants of transient expression was studiedwith regard to its decomplementing activity in complementconsumption-assays. However, no difference to control supernatantswithout CVF could be detected. Neither by a transient expression withthe serum replacement substance Nutridoma nor by culturing the cells inserum-free medium or by culturing in protein-free medium an activity ofthe recombinat CVF could be detected. The densitometrically quantifiedyields of expression with different replacement substances ranged from0.3-1.5 mg/l. Thus, they were slightly lower compared to the yieldsobtained by use of serum containing medium. The results indicated thatdirect characterization of rCVF in supernatants of transient expressionis not feasable. Consequently, further strategies where analyzed tocharacterize CVF and diverse hybrids after transient expression withoutthe need to apply time- and cost-consuming purification procedures forincreasing the concentration of C3/CVF-hybrid molecules. Differenttechniques for concentrating and purification were examined in order toachieve a separation of the components which prevent characterization ofrCVF in supernatants. Subsequent analyses of the samples in complementconsumption assays, however, did not reveal any success of the appliedstrategies. The decomplementing activity in the samples did not differsignificantly from corresponding controls, but the results pointed tothe present of high molecular weight compounds in the samplesinterfering with the complement consumption assays. Based on these data,solid-phase assays were developed for complement consumption analyses.One strategy employed a Strep-tag fused to the N-terminus of therecombinant proteins.

Hybrid H5 only comprises the functional relevant βchain of CVF andshares identity with human C3 of 90.7%. Based on the data available forhuman C3, it comprises the Factor B and Factor H-binding sites as wellas the cleavage sites for Factor I.

The hybrid was generated, fused with a Strep-tag and was successfullyexpressed in CHO-cells.

Subsequently, complement consuming activity was characterized in a solidphase-assay. It was confirmed that H5 has an activity comparable to CVF.

For the first time, a C3/CVF hybrid protein was provided where theCVF-portion could be reduced to 9% while retaining a CVF-like activity.The hybrid only comprises the CVFβ-chain and exhibits decomplementingactivity.

Up to now, the C3-region, which is homologous to the CVFβ-chain has onlyinsufficiently been studied. Therefore, only a few binding sites areidentified. A known binding site for the complement receptor CR3 islocated in the region of amino acids 1361-1381 (Wright et al., 1987).CR3, which is present on macrophages and killer cells, binds to C3biwhich has bound on the surface of pathogenes and mediates thedestruction of the pathogene (Newman et al., 1984). In the conducted invitro-assay, the binding of CR3 does not have any influence. In thevicinity of the CR3-region, the binding site for properdin with theamino acids 14241432 is in the analogous C3-region (Daoudaki et al.,1988. properdin binds and stabilizes the alternative C3-convertase C3bBb(Fearon et al., 1975). A binding site is also postulated in the CVFmolecule. The identity of the binding sites of C3 and CVF amounts to70%, which is clearly higher than the identity of the whole proteins(Fritznger et al., 1994). However, even an identity of 70% is inaccordance with crucial structural differences in the CVF-β-chain andthe C3-region homologous to the CVF-β-chain.

Considering the available data for human C3, H5 should be inactive. Itcomprises the human cleavage sites for Factor I and an additionalcleavage site for Factor H. Furthermore, the increased stability of theCVF-dependent convertase as a result of the stronger binding of CVF toFactor B, should be lost by humanization in H5 since all postulatedbinding regions for Factor B are located in regions replaced byC3-sequences. However, since no loss of activity was observed, it ispossible that structural differences between H5 and C3 protect thehybrid H5 from a regulation by Factor H and Factor I. Further, it wouldbe conceivable that an additional region which is located in the regionof the CVF-β-chain is responsible for the stronger binding to Factor B.

Quantification of hybrid H5 by antibody based procedures did not poseany problems. Since the identity with C3 amounts to approx. 91%, it canbe assumed that the polyclonal serum against C3 recognizes the hybrid H5with a comparable reactivity. Therefore, quantification based ondensitometric immunoblot analysis or ELISA with polyclonal anti-C3 serawas considered to provide reliable results.

This new molecule is of therapeutic relevance since it should clearlyexhibit a lower immunogenicity compared to CVF or CVF in which theα-chain is replaced by the corresponding human C3β-chain. Nevertheless,it shows the same complement-consuming activity. It should be possibleto apply this molecule in low concentrations.

After having demonstrated that hybrid H5 exerts complement activatingactivity, the analogous region of the human C3-cDNA was compared to thehomologous regions of the CVFcDNA. After analyzing the identities of CVFand human C3 in the analogous terminal regions, a further construct wasgenerated, H6. Hybrid H6 corresponds to human C3 in the first 1527 aminoacids; the C-terminus of the protein is CVF-sequence and exhibits anidentity of 96.3% to human C3.

After cloning, the hybrid was transiently expressed in CHO-cells andyields of 1-2 mg/l were obtained. Subsequent analysis of the activity ofthe hybrid H6 in a solid phase-assay demonstrated a substantialcomplement-consuming activity.

Upon quantification, activity decreased only by 50% when utilizingcomparable amounts of H6 and CVF in the solid phase-assay.

With hybrids H5 and H6, two humanized CVF-molecules were generated,which can be used in a therapeutic application. The hybrid H5 exhibits acomplement-consuming activity comparable to that of CVF. Hybrid H6 shows96% identity to human C3 and, most probably, this molecule is notimmunogenic. However, H6 shows a loss of approx. 50% in activitycompared to the CVF-molecule.

Since hybrid H6 could also be identified as a C3-derivative containingless than 4% foreign amino acid residues, further data were collected.First, a His-tag fusion protein was generated. Hybrid H6 was providedwith a His-tag using oligonucleotides as already done for the cloning ofthe Strep-tag. The His-tag fusion protein was successfully expressed inCHO-cells given yields of 1-3 mg/l. After generating a stable cell-line,hybrid His-H6 was enriched using IMAC. However, the majority of theprotein could not be immobilized and was detected in the flow-through.Additionally, the elution fractions were contaminated by a strongprotein background. Nevertheless, sufficient amounts of the protein wereobtained for further analyses.

The protein was densitometrically determined in the immunoblot andquantification was confirmed using immobilized anti-C3 antibodies in aSandwich-ELISA. Concentrations of 3-4 mg/l were determined. In asubsequent complement-consumption-assay, in which up to 80 ng proteinwas employed, a loss of activity of only 32% compared to CVF wasdemonstrated. The results show that the generated molecule exhibits aclear complement-consuming activity despite its 96% humanization.Therefore, the requirements of low dose applications are fulfilled.Therefore, CVF is an attractive complement modulator.

Additionally, the purified protein was employed in a BystanderLysis-Assay for the determination of fluid-phase-C5-convertase activity.In contrast to CVF, hybrid H6 did not exert significant fluidphase-C5-convertase activity. The Bystander Lysis-activity of CVF leadsto a fast and massive accumulation of C5a, which can cause severe tissuedamages (Till et al., 1982; Schmid et al., 1997). Therefore, a loss inC5-convertase activity caused by humanizing seems to represent anadvantage.

SUMMARY

Using cassette mutagenetic C3-derivatives were generated which arecapable of forming stable C3-convertases. Specific sequences of CVF wereutilized for the replacement of corresponding C3-regions (cf. alignmentin FIG. 1).

All C3/CVF hybrids as well as CVF and human C3 as such were utilized inparallel complement consumption assays.

The assays confirmed that the complement-consuming activity of both CVFand H5, the latter of which has 90.7% identity to human C3, arecomparable. The activity of H6 having 96% identity to human C3 wasslightly decreased in comparison to CVF (FIG. 16).

The therapeutic application of a complement modulator or inhibitor isattractive for treating several complement-associated diseases ordiseases affected by complement activation such as asthma (Regal et al.1993), systemic lupus erythematodes (Belmont et al., 1996),glomerulonephritis (Couser et al, 1985), rheumatoid arthritis (Kemp etal., 1992), Alzheimer's disease (Rogers et al., 1992), multiplesclerosis (Piddlesden et al., 1996), sepsis (Hack et al., 1989),hyperacute rejection and transplant rejection (Bach et al., 1995,Baldwin et al., 1995), cardiopulmonary bypass, myocardial infarction,angioplasty, nephritis, dermatomyositis, pemphigoid, spinal cord injury,and Parkinson's disease.

Comparison of complement inhibitors with respect to production costs anddosage requirements for therapeutic applications indicates a preferencefor the C3/CVF hybrid proteins. The generated C3-derivatives are, asenzymes, superior to other complement inhibitors. The 32% activity lossobserved for H6 can be compensated by a higher application dose ofapprox. 400 μg/kg. However, this dose is still low compared to otherinhibitors known in the art which require up to 80 fold higher doses tobe applied. The current invention provides human C3-derivatives that arecapable of forming C3-convertases exerting an extended CVF, Bb-likehalf-life of several hours, compared to 1.5 minutes of the naturallyoccurring C3-convertase, thus escaping the physiological degradationmechanisms. The high degrees of identity to human C3 should allowrepetitive therapeutic applications of the polypeptides of theinvention.

LIST OF ABBREVIATIONS

-   A Adenine-   ABTS 2,2′-Azino-bis(2-ethylbenzthiazoline-6-sulfonic acid)-   Amp Ampicillin resistance gene-   AP Alkaline phosphatase-   APS Ammonium persulfate-   BCIP 5-Bromo-4-chloro-3-indolylphosphate-   bp Base pairs-   BSA Bovine serum albumin-   C Cytosine-   C1-Inh C1 inhibitor-   C4bp C4 binding protein-   C3 Third complement protein-   CAPS 3-Cyclohexyl amine-1-propane sulfonic acid-   cDNA Complementary DNA-   CHO Chinese hamster ovary-   CIAP Calf intestinal alkaline phosphatase-   CMV Cytomegalovirus-   coC3 Cobra C3-   CR Complement receptor-   CVF Cobra Venom Factor-   DAF Decay accelerating factor-   ddH₂O Double destined water-   DMEM Dulbecco's Modified Eagle Medium-   DMF Dimethyl formamide-   DMSO Dimethyl sulfoxide-   DNA Deoxyribonucleic acid-   dNTP 2′-Deoxyribonucleic acid-   DTT Dithiothreitol-   EBV Epstein-Barr virus-   EDTA Ethylene diamine tetraacetate-   ELISA Enzyme-linked immunosorbent assay-   EK Enterokinase-   FCS Fetal calf serum-   G Guanine-   G418 Geneticin 418-   GFP Green fluorescent protein-   GMEM Glasgow Modified Eagle Medium-   GVBS⁺⁺ Veronal buffer with gelatin-   hC3 Human complement factor C3-   HEK Human embryonic kidney-   HEPES N-2-hydroxyethylpiperazine-N-2-ethylsulfonic acid-   His Histidine-   IMAC Immobilized metal ion affinity chromatography-   kb Kilo bases-   kDa Kilo dalton-   LB Luria-Bertani-   MAC Membrane attack complex-   MASP MBL-associated serine protease-   MBL Mannose-binding lectin-   MCP Membrane cofactor protein-   MCS Multiple cloning site-   NaAc Sodium acetate-   NBT Nitroblue tetrazolium chloride-   NEAA Non-essential amino acids-   NHS Normal human serum-   OD Optical density-   ori Origin of replication-   PAGE Polyacrylamide gel electrophoresis-   PBS Phosphate buffered saline-   PCR Polymerase chain reaction-   Penstrep Penicillin/streptomycin-   POD Peroxidase-   PVDF Polyvinylidene difluoride-   RNA Ribonucleic acid-   RNase Ribonuclease-   rpm Rotations per minute-   RT Room temperature-   SDS Sodium dodecyl sulfate-   St Strep-tagII-   T Thymine-   TAE Tris-acetate-EDTA buffer-   Taq Thermus aquaticus-   TBS Tris buffered saline-   TEMED N,N,N′,N′-Tetraethylmethylene diamine-   TES Tris EDTA sucrose-   TPBS PBS with Tween-   Tris Tris-(hydroxymethyl)-aminomethane-   TSS Transformation and storage solution-   Tween Polyoxyethylene sorbitane monolaurate-   U Unit-   VBS Veronal buffered saline-   VBS⁺⁺ Veronal buffered saline with MgCl₂ and CaCl₂-   v/v Volume per volume-   w/v Weight per volume-   xg multiple of gravitation

LIST OF REFERENCES

-   Alper, C. A.; Johnson, A. M.; Birtch, A. G. and Moore, F. D. (1969)    Human C′3: evidence for the liver as the primary site of synthesis.    Science, 163, 286-288.-   Ames, R. S.; Li, Y.; Sarau, H. M.; Nuthulaganti, P.; Foley, J. J.;    Ellis, C.; Zeng, Z.; Su, K.; Jurewicz, A, J.; Hertzberg, R. P.;    Bergsma, D. J. and Kumar, C. (1996) Molecular cloning and    characterization of the human anaphylatoxin C3a receptor J Biol    Chem, 271, 20231-20234.-   Bach, F. H.; Robson, S. C.; Winkler, H.; Ferran, C.; Stuhlmeier, K.    M.; Wrighton, C. J. and Hancock, W. W. (1995) Barriers to    xenotransplantation. Nat Med, 1, 869-873.-   Baldwin, W. M., 3rd; Pruitt, S. K.; Brauer, R. B.; Daha, M. R. and    Santilippo, F. (1995) Complement in organ transplantation.    Contributions to inflammation, injury, and rejection.    Transplantation, 59, 797-808.-   Ballow, M. and Cochrane, C. G. (1969) Two anticomplementary factors    in cobra venom: hemolysis of guinea pig erythrocytes to one of them.    J Immunol, 103, 944-952.-   Belmont, H. M.; Hopkins, P.; Edelson, H. S.; Kaplan, H. B.; Ludewig,    R.; Weissmann, G. and Abramson, S. (1986) Complement activation    during systemic lupus erythematosus. C3a and C5a anaphylatoxins    circulate during exacerbations of disease. Arthritis Rheum, 29,    1085-1089.-   Biesecker, G.; Dihel, L.; Enney, K. and Bendele, R. A. (1999)    Derivation of RNA aptamer inhibitors of human complement C5.    Immunopharmacology, 42, 219-230.-   Bohnsack, J. F. and Cooper, N. R. (1988) CR2 ligands modulate human    B cell activation. J Immunol, 141, 2569-2576.-   Burger, R.; Zilow, G.; Bader, A.; Friedlein, A. and Naser, W. (1988)    The C terminus of the anaphylatoxin C3a generated upon complement    activation represents a neoantigenic determinat with diagnostic    potential. J Immunol, 141, 553-558.-   Busch, K.; Piehler, J. and Fromm, H. (2000) Plant succinic    semialdehyde dehydrogenase: dissection of nucleotide binding by    surface plasmon resonance and fluorescence spectroscopy.    Biochemistry, 39, 10110-10117.-   Buyon, J. P.; Tamerius, J.; Ordorica, S.; Young, B. and    Abramson, S. B. (1992) Activation of the alternative complement    pathway accompanies disease flares in systemic lupus erythematosus    during pregnancy. Arthritis Rheum, 35, 55-61.-   Cheung, A. K.; Parker, C. J. and Hohnholt, M. (1994) Soluble    complement receptor type 1 inhibits complement activation induced by    hemodialysis membranes in vitro. Kidney Int, 46, 1680-1687.-   Christiansen, D.; Milland, J.; Thorley, B. R.; McKenzie, I. F. and    Loveland, B. E. (1996) A functional analysis of recombinant soluble    CD46 in vivo and a comparison with recombinant soluble forms of CD55    and CD35 in vitro. Eur J Immunol, 26, 578-585.-   Chrupcala, M.; Pomer, S.; Staehler, G.; Waldherr, R. and    Kirschfink, C. (1994) Prolongation of discordant renal xenograft    survival by depletion of complement. Comparative effects of    systemically administered cobra venom factor and soluble complement    receptor type 1 in a guinea-pig to rat model. Transpl Int, 7 Suppl    1, S650-653.-   Chrupcala, M.; Pomer, S.; Waldherr, R.; Staehler, G. and    Kirschfink, M. (1996) [Effect of complement modulation with the    soluble complement receptor sCR1 on survival and function of kidney    xenotransplant. An experimental study with a new guinea pig to rate    transplant model]. Urologe A, 35, 478-484.-   Cochrane, C. G.; Müller-Eberhard, H. J. and Aikin, B. S. (1970)    Depletion of plasma complement in vivo by a protein of cobra venom:    its effect on various immunologic reactions. J Immunol, 105, 55-69-   Cooper, P. D. (1985) Complement and cancer: activation of the    alternative pathway as a theoretical base for immunotherapy. Adv    Immun Cancer Ther, 1, 125-166.-   Cooper, P. D. and Sim, R. B. (1984) Substances that can trigger    activation of the alternative pathway of complement have    anti-melanoma activity in mice. Int J Cancer, 33, 683-687.-   Couser, W. G.; Baker, P. J. and Adler, S. (1985) Complement and the    direct mediation of immune glomerular injury: a new perspective.    Kidney Int, 28, 879-890.-   Couser, W. G.; Johnson, R. J.; Young, B. A.; Yeh, C. G.; Toth, C. A.    and Rudolph, A. R. (1995) The effects of soluble recombinant    complement receptor 1 on complement-mediated experimental    glomerulonephritis. J Am Soc Nephrol, 5, 1888-1894.-   Craddock, P. R.; Fehr, J.; Dalmasso, A. P; Brighan, K. L. and    Jacob, H. S. (1977) Hemodialysis leukopenia. Pulmonary vascular    leukostasis resulting from complement activation by dialyzer    cellophane membranes. J Clin Invest, 59, 879-888.-   Dalmasso, A. P. (1997) Role of complement in xenografts rejection,    in Xenotransplantation: The Transplantation of Organs and Tissues    Between Species, Vol. 2nd ed (Cooper, D. K.; Kemp, E.; Platt, J. L.    and White, D. J., eds), pp 33-60. Springer, Berlin.-   Daoudaki, M. E.; Becherer, J. D. and Lambris, J. D. (1988) A 3-4    amino acid peptide of the third component of complement mediates    properdin binding. J Immunol, 140, 1577-1580.-   Davies, A. (1996) Policing the membrane: cell surface proteins which    regulate complement. Res Immunol, 147, 82-87.-   Davis, A. E., 3rd and Harrison, R. A. (1982) Structural    characterization of factor I mediated cleavage of the third    component of complement. Biochemistry, 21, 5745-5749.-   DeBruijn, M. H. L. and Fey., G. H (1985) Human complement component    C3; cDNA coding sequence and derived primary structure. Proc. Natl.    Acad. Sci. USA, 708-712.-   Dolmer, K. and Sottrup-Jensen, L. (1993) Disulfide bridges in human    complement component C3b. FEBS Lett, 315, 85-90.-   Eldering, E.; Huijbregts, C. C.; Nuijens, J. H.; Verhoeven, A. J.    and Hack, C. E. (1993) Recombinant C1 inhibitor P5/P3 variants    display resistance to catalytic inactivation by stimulated    neutrophils. J Clin Invest, 91, 1035-1043.-   Elsner, J.; Oppermann, M.; Czech, W.; Dobos, G.; Schopf, E.;    Norgauer, J. and Kapp, A. (1994) C3a activates reactive oxygen    radical species production and intracellular calcium transients in    human eosinophils. Eur J Immunol, 24, 518-522.-   Eppinger, M. J.; Deeb, G. M.; Bolling, S. F. and Ward, P. A. (1997)    Mediators of ischemia-reperfusion injury of rat lung. Am J Pathol,    150, 1773-1784.-   Fearon, D. T. and Austen, K. F. (1975) Properdin: binding to C3b and    stabilization of the C3b-dependent C3-convertase. J Exp Med, 142,    856-863.-   Fecke, W.; Farries, T. C.; D'Cruz, L. G.; Napper, C. M. and    Harrison, R. A. (1998) Expression of factor I-resistant mutants of    the human complement component C3 in heterologous systems.    Xenotransplantation, 5, 29-34.-   Fiane, A. E.; Mollnes, T. E.; Videm, V.; Hovig, T.; Hogasen, K.;    Mellbye, O. J.; Spruce, L.; Moore, W. T.; Sahu, A. and    Lambris, J. D. (1999a) Prolongation of ex vivo-perfused pig    xenograft survival by the complement inhibitor Compstatin.    Transplant Proc, 31, 934-935.-   Fiane, A. E.; Mollnes, T. E.; Videm, V.; Hovig, T.; Hogasen, K.;    Mellbye, O. J.; Spruce, L.; Moore, W. T.; Sahu,A. and Lambris, J. D.    (1999b) Compstatin, a peptide inhibitor of C3, prolongs survival of    ex vivo perfused pig xenografts. Xenotransplantation, 6, 52-65.-   Fishelson, Z. (1991) Complement C3: a molecular mosaic of binding    sites. Mol Immunol, 28, 545-552.-   Fritzinger, D. C.; Bredehorst, R. and Vogel, C. W. (1994) Molecular    cloning and derived primary structure of cobra venom factor. Proc    Natl Acad Sci USA, 91, 12775-12779.-   Fritzinger, D. C.; Petrella, E. C.; Connelly, M. B.; Bredehorst, R.    and Vogel, C. W. (1992) Primary structure of cobra complement    component C3. J Immunol, 149, 3554-3562.-   Gonzalez-Rubio, C.; Ferreira-Cerdan, A.; Ponce, I. M.; Arpa, J.;    Fontan, G. and Lopez-Trascasa, M. (2001) Complement factor I    deficiency associated with recurrent menigitis coinciding with    menstruation. Arch Neurol, 58, 1923-1928.-   Gowda, D. C.; Petrella, E. C.; Raj, T. T.; Bredehorst, R. and    Vogel, C. W. (1994) Immunoreactivity and function of    oligosaccharides in cobra venom factor. J Immunol, 152, 2977-2986.-   Grier, A. H. and Vogel, C. W. (1989) The oligosaccharide chains of    cobra venom factor are required for complement activation. Mol    Immunol, 26, 563-574.-   Grier, A. H.; Schultz, M. and Vogel, C. W. (1987) Cobra venom factor    and human C3 share carbohydrate antigenicdeterminants. J Immunol,    139, 1245-1252.-   Griffiths, A. D.; Williams, S. C.; Hartley, O.; Tomlinson, I. M.;    Waterhouse, P.; Crosby, W. L.; Kontermann, R. E.; Jones, P. T.;    Low, N. M.; Allison, T. J. and et al. (1994) Isolation of high    affinity human antibodies directly from large synthetic repertoires.    Embo J, 13, 3245-3260.-   Gyongyossy-Issa, M. I.; McLeod, E. and Devine, D. V. (1994)    Complement activation in platelet concentrates is surface dependent    and modulated by the platelets. J Lab Clin Med, 123, 859-868.-   Hack, C. E.; Nuijens, J. H.; Felt-Bersma, R. J.; Schreuder, W. O.;    Eerenberg-Belmer, A. J.; Paardekooper, J.; Bronsveld, W. and    Thijs, L. G. (1989) Elevated plasma levels of the anaphylatoxins C3a    and C4a are associated with a fatal outcome in sepsis. Am J Med, 86,    20-26.-   Hack, C. E.; Voerman, H. J.; Eisele, B.; Keinecke, H. O.;    Nuijens, J. H.; Eerenberg, A. J.; Ogilvie, A.; Strack van    Schijndel, R. J.; Delvos, U. and Thijs, L. G. (1992) C1-esterase    inhibitor substitution in sepsis. Lancet, 339, 378.-   Heller, T.; Hennecke, M.; Baumann, U.; Gessner; J. E.; zu    Vilsendorf, A. M.; Baensch, M.; Boulay, F.; Kola, A.; Klos, A.;    Bautsch, W. and Kohl, J. (1999) Selection of a C5a receptor    antagonist from phage libraries attenuating the inflammatory    response in immune complex disease and ischemia/reperfusion injury.    J Immunol, 163, 985-994.-   Higgins, P. J.; Ko, J. L.; Lobell, R.; Sardonini, C.; Alessi, M. K.    and Yeh, C. G. (1997) A soluble chimeric complement inhibitory    protein that possesses both decay-accelerating and factor I cofactor    activities. J Immunol, 158, 2872-2881.-   Hirani, S.; Lambris, J. D. and Müller-Eberhard, H. J. (1986)    Structural analysis of the asparagine linked oligosaccharides of    human complement component C3. Biochem J, 233, 613-616.-   Homeister, J. W.; Satoh, P. and Lucchesi, B. R. (1992) Effects of    complement activation in the isolated heart. Role of the terminal    complement components. Circ Res, 71, 303-319.-   Horstick, G. (2002) C1-esterase inhibitor in ischemia and    reperfusion. Immunobiology, 205, 552-562.-   Kahnberg, K. E.; Lindhe, J. and Attstrom, R. (1976) The role of    complement in initial gingivitis. I. The effect of decomplementation    by cobra venom factor. J Periodontal Res, 11, 269-278.-   Kemp, E.; Dieperink, H.; Leth, P.; Jensenius, J. C.; Nielsen, B.;    Lillevang, S. T.; Salomon, S.; Steinbruchel, D., Larsen, S.;    Koch, C. and et al. (1994) Monoclonal antibodies to complement C3    prolong survival of discordant xenografts: guinea pig heart to rat    transplantation. Transplant Proc, 26, 1011-1015.-   Kemp, P. A.; Spragg, J. H.; Brown, J. C.; Morgan, B. P.; Gunn, C. A.    and Taylor, P. W. (1992) Immunohistochemical determination of    complement activation in joint tissues of patients with rheumatoid    arthritis and osteoarthritis using neoantigen-specific monoclonal    antibodies, Clin Lab Immunol, 37, 147-162.-   Kilgore, K. S.; Friedrichs, G. S.; Homeister, J. W. and    Lucchesi, B. R. (1994) The complement system in myocardial    ischaemia/reperfusion injury. Cardiovasc Res, 28, 437-444.-   Kinoshita, T.; Medof, M. E.; Silber, R. and Nussenzweig, V. (1985)    Distribution of decay-accelerating factor in the peripheral blood of    normal individuals and patients with paroxysmal nocturnal    hemoglobinuria. J Exp Med, 162, 75-92.-   Kirklin, J. K.; Westaby, S.; Blackstone, E. H.; Kirklin, J. W.;    Chenoweth, D. E. and Pacifico, A. D. (1983) Complement and the    damaging effects of cardiopulmonary bypass. J Thorac Cardiovasc    Surg, 86, 845-857.-   Kock, M. A. (1996) Expression and characterization of recombinant    cobra venom factor, Dissertation, Fachbereich Chemie. Universität    Hamburg, Hamburg.-   Kölln, J. Ziegelmüller, P.; Klensang, K.; Schneider, I.;    Bredehorst, R. and Andrä, J. (2001) Transient expression of active    Cobra Venom Factor (CVF) and CVF/C3 chimeras in mammalian cells.    Biol Chem, 382, 164.-   Konteatis, Z. D.; Siciliano, S. J.; Van Riper, G.; Molineaux, C. J.;    Pandya, S.; Fischer, P.; Rosen, H.; Mumford, R. A. and    Springer, M. S. (1994) Development of C5a receptor antagonists.    Differential loss of functional responses. J Immunol, 153,    4200-4205.-   Lachmann, P. J.; Pangburn, M. K. and Oldroyd, R. G. (1982) Breakdown    of C3 after complement activation. Identification of a new fragment    C3g, using monoclonal antibodies. J Exp Med, 156, 205-216.-   Laemmli, U. K. (1970) Cleavage of structural proteins during the    assembly of the head of bacteriophage T4. Nature, 227, 680-685.-   Law, S. K. and Dodds, A. W. (1997) The internal thioester and the    covalent binding properties of the complement proteins C3 and C4.    Protein Sci, 6, 263-274.-   Lennon, V. A.; Seybold, M. E.; Lindstrom, J. M.; Cochrane, C. and    Ulevitch, R. (1978) Role of complement in the pathogenesis of    experimental autoimmune myasthenia gravis. J Exp Med, 147, 973-983.-   Lens, J. W.; van den Berg, W. B.; van de Putte, L. B.; Berden, J. H.    and Lems, S. P. (1984) Flare-up of antigen-induced arthritis in mice    after challenge with intravenous antigen: effects of pretreatment    with cobra venom factor and anti-lymphocyte serum. Clin Exp Immunol,    57, 520-528.-   Lin, Y.; Soares, M. P.; Sato, K.; Csizmadia, E.; Robson, S. C.;    Smith, N. and Bach, F. H. (2000) Long-term survival of hamster    hearts in presensitized rats. J Immunol, 164, 4883-4892.-   Liszewski, M. K. and Atkinson, J. P. (1992) Membrane cofactor    protein. Curr Top Microbiol Immunol, 178, 45-60.-   Lublin, D. M. and Atkinson, J. P. (1989) Decay-accelerating factor:    biochemistry, molecular biology, and function. Annu Rev Immunol, 7,    35-58.-   Lublin, D. M. and Atkinson, J. P. (1990) Decay-accelerating factor    and membrane cofactor protein. Curr Top Microbiol Immunol, 153,    123-145.-   Lupton, S. and Levine, A. J. (1985) Mapping genetic elements of    Epstein-Barr virus that facilitate extrachromosomal persistence of    Epstein-Barr virus-derived plasmids in human cells. Mol Cell Biol,    5, 2533-2542.-   Makrides, S. C.; Scesney, S. M.; Ford, P. J.; Evans, K. S.;    Carson, G. R. and Marsh, H. C., Jr. (1992) Cell surface expression    of the C3b/C4b receptor (CR1) protects Chinese hamster ovary cells    from lysis by human complement. J. Biol Chem, 267, 24754-24761.-   Medicus, R. G.; Götze, O. and Müller-Eberhard, H. J. (1976)    Alternative pathway of complement: recruitment of precursor    properdin by the labile C3/C5 convertase and the potentiation of the    pathway. J Exp Med, 144, 1076-1093.-   Mollnes, T. E. (1997) Biocompatibility: complement as mediator of    tissue damage and as indicator of incompatibility. Exp Clin    Immunogenet, 14, 24-29.-   Mollnes, T. E. and Lachmann, P. J. (1988) Regulation of complement.    Scand J Immunol, 27, 127-142.-   Moran, P.; Beasley, H.; Gorrell, A.; Martin, E.; Gribling, P.;    Fuchs, H.; Gillett, N.; Burton, L. E. and Caras, I. W. (1992) Human    recombinant soluble decay accelerating factor inhibits complement    activation in vitro and in vivo. J Immunol, 149, 1736-1743.-   Morariu, M. A. and Dalmasso, A. P. (198) Experimental allergic    encephalomyelitis in cobra venom factor-treated and C4-deficient    guinea pigs. Ann Neurol, 4, 427-430.-   Morgan, B. P. and Walport, M. J. (1991) Complement deficiency and    disease. Immunol Today, 12, 301-306.-   Morgan, B. P.; Gasque, P.; Singhrao, S. K. and    Piddlesden, S. J. (1997) Role of complement in inflammation and    injury in the nervous system. Exp Clin Immunogenet, 14, 19-23.-   Müller-Eberhard, H. J. and Fjellstrom, K. E. (1971) Isolation of the    anticomplementary protein from cobs venom and its mode of action on    C3. J Immunol, 107, 1666-1672.-   Newman, S. L.; Devery-Pocius, J. E.; Ross, G. D. and    Henson, P. M. (1984) Phagocytosis by human monocytederived    macrophages. Independent function of receptors for C3b (CR1) and    iC3b (CR3). Complement, 1, 213-227.-   Oberholzer, J.; Yu, D.; Triponez, F.; Cretin, N.; Andereggen, E.;    Mentha, G.; White, D.; Buehler, L.; Morel, P. and Lou, J. (1999)    Decomplementation with cobra venom factor prolongs survival of    xenografted islets in a rat to mouse model. Immunology, 97, 173-180.-   O'Keefe, M. C.; Caporale, L. H. and Vogel, C. W. (1988) A novel    cleavage product of human complement component C3 with structural    and functional properties of cobra venom factor. J Biol Chem, 263,    12690-12697.-   Oran, A. E. and Isenman, D. E. (1999) Identification of residues    within the 727-767 segment of human complement component C3    important for its interaction with factor H and with complement    receptor 1 (CR1, CD35). J Biol Chem, 274, 5120-5130.-   Pang, A. S. and Minta, J. 0. (1980) Inhibition of vitamin D2-induced    arteriosclerosis in rats by depletion of complement with cobra venom    factor. Artery, 7, 109-122.-   Pangburn, M. K. and Müller-Eberhard, H. J. (1984) The alternative    pathway of complement. Springer Semin Immunopathol, 7, 163-192.-   Park, K. W.; Tofukuji, M.; Metais, C.; Comunale, M. E.; Dai, H. B.;    Simons, M.; Stahl, G. L.; Agah, A. and Sellke, F. W. (1999)    Attenuation of endothelium-dependent dilation of pig pulmonary    arterioles after cardiopulmonary bypass is prevented by monoclonal    antibody to complement C5a. Anesth Analg, 89, 42-48.-   Pellas, T. C.; Boyar, W.; van Oostrum, J.; Wasvary, J.; Fryer, L.    R.; Pastor, G.; Sills, M.; Braunwalder, A.; Yarwood, D. R.; Kramer,    R.; Kimble, B.; Hadala, J.; Haston, W.; MoreiraLudewig, R.;    Uziel-Fusi, S.; Peters, P.; Bill, K. and Wennogle, L. P. (1998)    Novel C5a receptor antagonists regulate neutrophil functions in    vitro and in vivo. J Immunol, 160, 5616-5621.-   Piddlesden, S. J.; Jiang, S.; Levin, J. L.; Vincent, A. and    Morgan, B. P. (1996) Soluble complement receptor 1 (sCR1) protects    against experimental autoimmune myasthenia gravis. J Neuroimmunol,    71, 173-177.-   Piddlesden, S. J.; Storch, M. K.; Hibbs, M.; Freeman, A. M.;    Lassmann, H. and Morgan, B. P. (1994) Soluble recombinat complement    receptor 1 inhibits inflammation and demyelination in antibody    mediated demyelinating experimental allergic encephalomyelitis. J    Immunol, 152, 5477-5484.-   Pinter, C.; Siccardi, A, G.; Lopalco, L.; Longhi, R. and    Clivio, A. (1995) HIV glycoprotein 41 and complement factor H    interact with each other and share functional as well as antigenic    homology. AIDS Res Hum Retroviruses, 11, 971-980.-   Regal, J. F. and Fraser, D. G. (1996) Systemic complement system    depletion does not inhibit cellular accumulation in antihistamine    pretreated allergic guinea pig lung. Int Arch Allergy Immunol, 109,    150-160.-   Regal, J. F.; Fraser, D. G. and Toth, C. A. (1993) Role of the    complement system in antigen-induced bronchoconstriction and changes    in blood pressure in the guinea pig. J Pharmacol Exp Ther, 267,    979-988.-   Rogers, J.; Cooper, N. R.; Webster, S.; Schultz, J.; McGeer, P. L.;    Styren, S. D.; Civin, W. H.; Brachova, L.; Bradt, B.; Ward, P. and    et al. (1992) Complement activation by beta-amyloid in Alzheimer    disease. Proc Natl Acad Sci USA, 89, 10016-10020.-   Ross, S. C. and Densen, P. (1984) Complement deficiency states and    infection; epidemiology, pathogenesis and consequences of neisserial    and other infections in an immune deficiency. Medicine (Baltimore),    63, 243-273.-   Saiki, R. K.; Gelfand, D. H.; Stoffel, S,; Scharf, S. J.; Higuchi,    R.; Horn, G. T.; Mullis, K. B. and Erlich, H. A. (1988) Primer    directed enzymatic amplification of DNA with a thermostable DNA    polymerase. Science, 239, 487-491.-   Scharfstein, J.; Ferreira, A.; Gigli, I. and Nussenzweig, V. (1978)    Human C4-binding protein. I. Isolation and characterization. J Exp    Med, 148, 207-222.-   Schmid, E.; Warner, R. L.; Crouch, L. D.; Friedl, H. P.; Till, G.    O.; Hugli, T. E. and Ward, P. A. (1997) Neutrophilchemotactic    activity and C5a following systemic activation of complement in    rats. Inflammation, 21, 325-333.-   Schmidt, T. G.; Koepke, J.; Frank, R. and Skerra, A. (1996)    Molecular interaction between the Strep-tag affinity peptide and its    cognate target, streptavidin. J Mol Biol, 255, 753-766.-   Sim, R. B.; Reboul, A.; Arlaud, G. J.; Villiers, C. L. and    Colomb, M. G. (1979) Interaction of 125I-labelled complement    subcomponents C-1r and C-1s with protease inhibitors in plasma. FEBS    Lett, 97, 111-115.-   Skerra, A. and Schmidt, T. G. (2000) Use of the Strep-Tag and    streptavidin for detection and purification of recombinant proteins.    Methods Enzymol, 326, 271-304.-   Smyth, N.; Odenthal, U.; Merkl, B. and Paulsson, M. (2000)    Eukaryotic expression and purification of recombinant extracellular    matrix proteins carrying the Strep II tag. Methods Mol Biol, 139,    49-57.-   Spillner, E. (2002) Selektion und Expression von rekombinanten    Antikörpern für analytische und therapeutische Applikationen, in    Fachbereich Chemie. Universität Hamburg, Hamburg.-   Stoiber, H.; Schneider, R.; Janatova, J. and Dierich, M. P. (1995)    Human complement proteins C3b, C4b, factor H and properdin react    with specific sites in gp120 and gp41, the envelope proteins of    HI-Vl. Immunobiology, 193, 98-113.-   Struber, M.; Hagl, C.; Hirt, S. W.; Cremer, J.; Harringer, W. and    Haverich, A. (1999) C1-esterase inhibitor in graft failure after    lung transplantation. Intensive Care Med, 25, 1315-1318.-   Sugita, Y.; Ito, K.; Shiozuka, K.; Suzuki, H.; Gushima, H.;    Tomita M. and Masuho, Y. (1994) Recombinant soluble CD59 inhibits    reactive hemolysis with complement. Immunology, 82, 34-41.-   Tack, B. F.; Harrison, R. A.; Janatova, J.; Thomas, M. L. and    Prahl, J. W. (1980) Evidence for presence of an internal thiolester    bond in third component of human complement. Proc Natl Acad Sci USA,    77, 5764-5768.-   Taniguchi, S.; Kobayashi, T.; Neethling, F. A.; Ye, Y.; Niekrasz,    M.; White, D. T. and Cooper, D. K. (1996) Cobra venom factor    stimulates anti-alpha-galactose antibody production in baboons.    Implications for pig-to-human xenotransplantation. Transplantation,    62, 678-681.-   Taniguchi-Sidle, A. and Isenman, D. E. (1994) Interactions of human    complement component C3 with factor B and with complement receptors    type 1 (CR1, CD35) and type 3 (CR3, CD11b/CD18) involve an acidic    sequence at the N-terminus of C3 alpha′-chain. J Immunol, 153,    5285-5302.-   Till, G. O.; Johnson, K. J.; Kunkel, R. and Ward, P. A. (1982)    Intravascular activation of complement and acute lung injury.    Dependency on neutrophils and toxic oxygen metabolites. J Clin    Invest, 69, 1126-1135.-   Vakeva, A. P.; Agah, A.; Rollins, S. A.; Matis, L. A.; Li, L. and    Stahl, G. L. (1998) Myocardial infarction and apoptosis after    myocardial ischemia and reperfusion: role of the terminal complement    components and inhibition by anti-C5 therapy. Circulation, 97,    2259-2267.-   Vogel, C. W. (1985) Untersuchungen zur Strukturhomologie von    Kobrafaktor mit dem menschlichen Komplementprotein C3 sowie Synthese    kovalenter Hybridproteine aus Kobnfaktor und monoklonalen    Antikörpern als selektiv-zytolytisches Prinzip, Dissertation,    Fachbereich Chemie. Universität Hamburg, Hamburg.-   Vogel, C. W. and Müller-Eberhard, H. J. (1982) The cobra venom    factor-dependent C3-convertase of human complement. A kinetic and    thermodynamic analysis of a protease acting on its natural high    molecular weight substrate. J Biol Chem, 257, 8292-8299.-   Vogel, C. W. and Müller-Eberhard, H. J. (1984) Cobra venom factor:    improved method for purification and biochemical characterization. J    Immunol Methods, 73, 203-220,-   Vogel, C. W.; Smith, C. A. and Müller-Eberhard, H. J. (1984) Cobra    venom factor: structural homology with the third component of human    complement. J Immunol, 133, 3235-3241.-   Vogel, C. W.; Wilkie, S. D. and Morgan, A. C. (1985) In vivo studies    with covalent conjugates of cobra venom factor and monoclonal    antibodies to human tumors. Haematol Blood Transfus, 29, 514-517.-   Vogel, C. W.; Bredehorst, R.; Fritzinger, D. C.; Grunwald, T.;    ZiegelMüller, P. and Kock, M. A. (1996) Structure and function of    cobra venom factor, the complement-activating protein in cobra    venom. Adv Exp Med Biol, 391, 97-114.-   Volanakis, J. E. (1995) Transcriptional regulation of complement    genes. Annu Rev Immunol, 13, 277-305.-   Wang, Y.; Rollins, S. A.; Madri, J. A. and Matis, L. A. (1995) Anti    CS monoclonal antibody therapy prevents collagen-induced arthritis    and ameliorates established disease. Proc Natl Acad Sci USA, 92,    8955-8959.-   Wang, Y.; Hu, Q.; Madri; J. A.; Rollins, S. A.; Chodera, A and    Matis, L. A. (1996) Amelioration of lupus like autoimmune disease in    NZB/WF1 mice after treatment with a blocking monoclonal antibody    specific for complement component C5. Proc Natl Acad Sci USA, 93,    8563-8568.-   Wehrhahn, D. (2000) Untersuchungen zur Struktur-Funktionsbeziehungen    von Kobra Venom Faktor-Konstruktion und rekombinante Expression von    Kobra Venom Faktor/Kobra C3 Hybriden, Dissertation, Fachbereich    Chemie. Universität Hamburg, Hamburg.-   Weisman, H. F.; Bartow, T.; Leppo, M. K.; Marsh, H. C., Jr.;    Carson, G. R.; Concino, M. F.; Boyle, M. P.; Roux, K. H.;    Weisfeldt, M. L. and Fearon, D. T. (1990) Soluble human complement    receptor type 1: in vivo inhibitor of complement suppressing post    ischemic myocardial inflammation and necrosis. Science, 249,    146-151.-   Whiss, P. A. (2002) Pexelizumab Alexion. Curr Opin Investig Drugs,    3, 870-877.-   Williams, K. C.; Ulvestad, E. and Hickey, W. F. (1994) Immunology of    multiple sclerosis. Clin Neurosci, 2, 229-245.-   Wright, S.D.; Reddy, P. D.; Jong, M. T. and Erickson, B. W. (1987)    C3bi receptor (complement receptor type 3) recognizes a region of    complement protein C3 containing sequence Arg-Gly-Asp. Proc Natl    Acad Sci USA, 84, 1965-8.-   Zimmerman, J. L.; Dellinger, R. P.; Straube, R. C. and    Levin, J. L. (2000) Phase I trial of the recombinant soluble    complement receptor 1 in acute lung injury and acute respiratory    distress syndrome. Crit Care Med, 28, 3149-3154.-   Zipfel, P. F.; Skerka, C.; Caprioli, U.; Manuelian, T.; Neumann, H.    H.; Noris, M. and Remuzzi, G. (2001) Complement factor H and    hemolytic uremic syndrome, Int Immunopharmacol, 1, 461-468.

We claim:
 1. A method for decomplementation in a human subjectcomprising administering an effective amount of a hybrid proteincomprising a partial sequence of human complement component C3 (humanC3), the human C3 having the amino acid sequence set forth as SEQ ID NO:2, and a partial sequence of Cobra Venom Factor (CVF), the CVF havingthe amino acid sequence set forth as SEQ ID NO: 4, wherein the carboxyterminal part of at least 68 amino acids of said human C3 is replaced bythe partial sequence of CVF; wherein the partial sequence of CVFcomprises at least 68 carboxy terminal amino acids of CVF, and whereinsaid protein has at least 90 percent identity to said human C3; andwherein said protein is capable of forming a stable C3 convertase. 2.The method of claim 1 wherein the hybrid protein has an amino acidsequence selected from the group consisting of SEQ ID Nos: 6, 8, 10, and12.
 3. A method for treating a patient suffering from acomplement-associated disorder or a disorder affected by complementactivation comprising administering an effective amount of a hybridprotein comprising a partial sequence of human complement component C3(human C3), the human C3 having the amino acid sequence set forth as SEQID NO: 2, and a partial sequence of Cobra Venom Factor (CVF), the CVFhaving the amino acid sequence set forth as SEQ ID NO: 4, wherein thecarboxy terminal part of at least 68 amino acids of said human C3 isreplaced by the partial sequence of CVF; wherein the partial sequence ofCVF comprises at least 68 carboxy terminal amino acids of CVF, andwherein said protein has at least 90 percent identity to said human C3;and wherein said protein is capable of forming a stable C3 convertase.4. The method of claim 3, wherein said complement-associated disorder orsaid disorder affected by complement activation is selected from thegroup consisting of asthma, systemic lupus erythematodes,glomerulonephritis, rheumatoid arthritis, Alzheimer's disease, multiplesclerosis, myocardial ischemia, reperfusion, sepsis, hyperacuterejection, transplant rejection, cardiopulmonary bypass, myocardialinfarction, angioplasty, nephritis, dermatomyositis, pemphigoid, spinalcord injury and Parkinson's disease.
 5. A method for treating a patientsuffering from a complement-associated disorder or a disorder affectedby complement activation comprising administering an effective amount ofa hybrid protein comprising a partial sequence of human complementcomponent C3 (human C3), the human C3 having the amino acid sequence setforth as SEQ ID NO: 2, and a partial sequence of Cobra Venom Factor(CVF), the CVF having the amino acid sequence set forth as SEQ ID NO: 4,wherein the carboxy terminal part of at least 68 amino acids of saidhuman C3 is replaced by the partial sequence of CVF; wherein the partialsequence of CVF comprises at least 68 carboxy terminal amino acids ofCVF, and wherein said protein has at least 90 percent identity to saidhuman C3; wherein said protein is capable of forming a stable C3convertase; and wherein the hybrid protein has an amino acid sequenceselected from the group consisting of SEQ ID Nos: 6, 8, 10, and
 12. 6.The method of claim 5, wherein said complement-associated disorder orsaid disorder affected by complement activation is selected from thegroup consisting of asthma, systemic lupus erythematodes,glomerulonephritis, rheumatoid arthritis, Alzheimer's disease, multiplesclerosis, myocardial ischemia, reperfusion, sepsis, hyperacuterejection, transplant rejection, cardiopulmonary bypass, myocardialinfarction, angioplasty, nephritis, dermatomyositis, pemphigoid, spinalcord injury, or Parkinson's disease.
 7. The method of claim 1 whereinthe hybrid protein is in a composition.
 8. The method of claim 1 whereinthe hybrid protein has at least 95 percent identity to said human C3.